Combinations and modes of administration of therapeutic agents and combination therapy

ABSTRACT

The present invention provides combination therapy methods of treating proliferative diseases (such as cancer) comprising a first therapy comprising administering to an individual an effective amount of a taxane in a nanoparticle composition, and a second therapy which may include, for example, radiation, surgery, administration of chemotherapeutic agents (such as an anti-VEGF antibody), or combinations thereof. Also provided are methods of administering to an individual a drug taxane in a nanoparticle composition based on a metronomic dosing regime.

TECHNICAL FIELD

The present invention relates to methods and compositions for thetreatment of proliferative diseases comprising the administration of acombination of a taxane and at least one other and other therapeuticagents, as well as other treatment modalities useful in the treatment ofproliferative diseases. In particular, the invention relates to the useof nanoparticles comprising paclitaxel and albumin (such as Abraxane®)in combination with other chemotherapeutic agents or radiation, whichmay be used for the treatment of cancer.

BACKGROUND

The failure of a significant number of tumors to respond to drug and/orradiation therapy is a serious problem in the treatment of cancer. Infact, this is one of the main reasons why many of the most prevalentforms of human cancer still resist effective chemotherapeuticintervention, despite certain advances in the field of chemotherapy.

Cancer is now primarily treated with one or a combination of three typesof therapies: surgery, radiation, and chemotherapy. Surgery is atraditional approach in which all or part of a tumor is removed from thebody. Surgery generally is only effective for treating the earlierstages of cancer. While surgery is sometimes effective in removingtumors located at certain sites, for example, in the breast, colon, andskin, it cannot be used in the treatment of tumors located in otherareas, inaccessible to surgeons, nor in the treatment of disseminatedneoplastic conditions such as leukemia. For more than 50% of cancerindividuals, by the time they are diagnosed they are no longercandidates for effective surgical treatment. Surgical procedures mayincrease tumor metastases through blood circulation during surgery. Mostof cancer individuals do not die from the cancer at the time ofdiagnosis or surgery, but rather die from the metastasis and therecurrence of the cancer.

Other therapies are also often ineffective. Radiation therapy is onlyeffective for individuals who present with clinically localized diseaseat early and middle stages of cancer, and is not effective for the latestages of cancer with metastasis. Radiation is generally applied to adefined area of the subject's body which contains abnormal proliferativetissue, in order to maximize the dose absorbed by the abnormal tissueand minimize the dose absorbed by the nearby normal tissue. However, itis difficult (if not impossible) to selectively administer therapeuticradiation to the abnormal tissue. Thus, normal tissue proximate to theabnormal tissue is also exposed to potentially damaging doses ofradiation throughout the course of treatment. There are also sometreatments that require exposure of the subject's entire body to theradiation, in a procedure called “total body irradiation”, or “TBI.” Theefficacy of radiotherapeutic techniques in destroying abnormalproliferative cells is therefore balanced by associated cytotoxiceffects on nearby normal cells. Because of this, radiotherapy techniqueshave an inherently narrow therapeutic index which results in theinadequate treatment of most tumors. Even the best radiotherapeutictechniques may result in incomplete tumor reduction, tumor recurrence,increasing tumor burden, and induction of radiation resistant tumors.

Chemotherapy involves the disruption of cell replication or cellmetabolism. Chemotherapy can be effective, but there are severe sideeffects, e.g., vomiting, low white blood cells (WBC), loss of hair, lossof weight and other toxic effects. Because of the extremely toxic sideeffects, many cancer individuals cannot successfully finish a completechemotherapy regime. Chemotherapy-induced side effects significantlyimpact the quality of life of the individual and may dramaticallyinfluence individual compliance with treatment. Additionally, adverseside effects associated with chemotherapeutic agents are generally themajor dose-limiting toxicity (DLT) in the administration of these drugs.For example, mucositis is one of the major dose limiting toxicity forseveral anticancer agents, including the antimetabolite cytotoxic agents5-FU, methotrexate, and antitumor antibiotics, such as doxorubicin. Manyof these chemotherapy-induced side effects if severe may lead tohospitalization, or require treatment with analgesics for the treatmentof pain. Some cancer individuals die from the chemotherapy due to poortolerance to the chemotherapy. The extreme side effects of anticancerdrugs are caused by the poor target specificity of such drugs. The drugscirculate through most normal organs of individuals as well as intendedtarget tumors. The poor target specificity that causes side effects alsodecreases the efficacy of chemotherapy because only a fraction of thedrugs is correctly targeted. The efficacy of chemotherapy is furtherdecreased by poor retention of the anti-cancer drugs within the targettumors.

Due to the severity and breadth of neoplasm, tumor and cancer, there isa great need for effective treatments of such diseases or disorders thatovercome the shortcomings of surgery, chemotherapy, and radiationtreatment.

Problems of Chemotherapeutic Agents

The drug resistance problem is a reason for the added importance ofcombination chemotherapy, as the therapy both has to avoid the emergenceof resistant cells and to kill pre-existing cells which are already drugresistant.

Drug resistance is the name given to the circumstance when a diseasedoes not respond to a treatment drug or drugs. Drug resistance can beeither intrinsic, which means the disease has never been responsive tothe drug or drugs, or it can be acquired, which means the disease ceasesresponding to a drug or drugs that the disease had previously beenresponsive to. Multidrug resistance (MDR) is a specific type of drugresistance that is characterized by cross-resistance of a disease tomore than one functionally and/or structurally unrelated drugs.Multidrug resistance in the field of cancer is discussed in greaterdetail in “Detoxification Mechanisms and Tumor Cell Resistance toAnticancer Drugs,” by Kuzmich and Tew, particularly section VII “TheMultidrug-Resistant Phenotype (MDR),” Medical Research Reviews, Vol. 11,No. 2, 185-217, (Section VII is at pp. 208-213) (1991); and in“Multidrug Resistance and Chemosensitization: Therapeutic Implicationsfor Cancer Chemotherapy,” by Georges, Sharom and Ling, Advances inPharmacology, Vol. 21, 185-220 (1990).

One form of multi-drug resistance (MDR) is mediated by a membrane bound170-180 kD energy-dependent efflux pump designated as P-glycoprotein(P-gp). P-glycoprotein has been shown to play a major role in theintrinsic and acquired resistance of a number of human tumors againsthydrophobic, natural product drugs. Drugs that act as substrates for andare consequently detoxified by P-gp include the vinca alkaloids(vincristine and vinblastine), anthracyclines (Adriamycin), andepipodophyllotoxins (etoposide). While P-gp associated MDR is a majordeterminant in tumor cell resistance to chemotherapeutic agents, it isclear that the phenomenon of MDR is multifactorial and involves a numberof different mechanisms.

A major complication of cancer chemotherapy and of antiviralchemotherapy is damage to bone marrow cells or suppression of theirfunction. Specifically, chemotherapy damages or destroys hematopoieticprecursor cells, primarily found in the bone marrow and spleen,impairing the production of new blood cells (granulocytes, lymphocytes,erythrocytes, monocytes, platelets, etc.). Treatment of cancerindividuals with 5-fluorouracil, for example, reduces the number ofleukocytes (lymphocytes and/or granulocytes), and can result in enhancedsusceptibility of the individuals to infection. Many cancer individualsdie of infection or other consequences of hematopoietic failuresubsequent to chemotherapy. Chemotherapeutic agents can also result insubnormal formation of platelets which produces a propensity towardhemorrhage. Inhibition of erythrocyte production can result in anemia.For some cancer individuals, the risk of damage to the hematopoieticsystem or other important tissues frequently limits the opportunity forchemotherapy dose escalation of chemotherapy agents high enough toprovide good antitumor or antiviral efficacy. Repeated or high dosecycles of chemotherapy may be responsible for severe stem cell depletionleading to serious long-term hematopoietic sequelea and marrowexhaustion.

Prevention of, or protection from, the side effects of chemotherapywould be a great benefit to cancer individuals. For life-threateningside effects, efforts have concentrated on altering the dose andschedules of the chemotherapeutic agent to reduce the side effects.Other options are becoming available, such as the use of granulocytecolony stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF),epidermal growth factor (EGF), interleukin 11, erythropoietin,thrombopoietin, megakaryocyte development and growth factor, pixykines,stem cell factor, FLT-ligand, as well as interleukins 1, 3, 6, and 7, toincrease the number of normal cells in various tissues before the startof chemotherapy (See Jimenez and Yunis, Cancer Research 52:413-415;1992). The mechanisms of protection by these factors, while not fullyunderstood, are most likely associated with an increase in the number ofnormal critical target cells before treatment with cytotoxic agents, andnot with increased survival of cells following chemotherapy.

Chemotherapeutic Targeting For Tumor Treatment

Both the growth and metastasis of solid tumors areangiogenesis-dependent (Folkman, J. Cancer Res., 46, 467-73 (1986);Folkman, J. Nat. Cancer Inst., 82, 4-6 (1989); Folkman et al., “TumorAngiogenesis,” Chapter 10, pp. 206-32, in The Molecular Basis of Cancer,Mendelsohn et al., eds. (W. B. Saunders, 1995)). It has been shown, forexample, that tumors which enlarge to greater than 2 mm in diameter mustobtain their own blood supply and do so by inducing the growth of newcapillary blood vessels. After these new blood vessels become embeddedin the tumor, they provide nutrients and growth factors essential fortumor growth as well as a means for tumor cells to enter the circulationand metastasize to distant sites, such as liver, lung or bone (Weidner,New Eng. J. Med., 324(1), 1-8 (1991)). When used as drugs intumor-bearing animals, natural inhibitors of angiogenesis can preventthe growth of small tumors (O'Reilly et al., O'Reilly et al., Cell, 79,315-28 (1994)). Indeed, in some protocols, the application of suchinhibitors leads to tumor regression and dormancy even after cessationof treatment (O'Reilly et al., Cell, 88, 277-85 (1997)). Moreover,supplying inhibitors of angiogenesis to certain tumors can potentiatetheir response to other therapeutic regimes (e.g., chemotherapy) (see,e.g., Teischer et al., Int. J. Cancer, 57, 920-25 (1994)).

Protein tyrosine kinases catalyze the phosphorylation of specifictyrosyl residues in various proteins involved in the regulation of cellgrowth and differentiation (A. F. Wilks, Progress in Growth FactorResearch, 1990, 2, 97-111; S. A. Courtneidge, Dev. Supp.l, 1993, 57-64;J. A. Cooper, Semin. Cell Biol., 1994, 5(6), 377-387; R. F. Paulson,Semin. Immunol., 1995, 7(4), 267-277; A. C. Chan, Curr. Opin. Immunol.,1996, 8(3), 394-401). Protein tyrosine kinases can be broadly classifiedas receptor (e.g. EGFr, c-erbB-2, c-met, tie-2, PDGFr, FGFr) ornon-receptor (e.g. c-src, Ick, Zap70) kinases. Inappropriate oruncontrolled activation of many of these kinases, i.e. aberrant proteintyrosine kinase activity, for example by over-expression or mutation,has been shown to result in uncontrolled cell growth. For example,elevated epidermal growth factor receptor (EGFR) activity has beenimplicated in non-small cell lung, bladder and head and neck cancers,and increased c-erbB-2 activity in breast, ovarian, gastric andpancreatic cancers. Thus, inhibition of protein tyrosine kinases shouldbe useful as a treatment for tumors such as those outlined above.

Growth factors are substances that induce cell proliferation, typicallyby binding to specific receptors on cell surfaces. Epidermal growthfactor (EGF) induces proliferation of a variety of cells in vivo, and isrequired for the growth of most cultured cells. The EGF receptor is a170-180 kD membrane-spanning glycoprotein, which is detectable on a widevariety of cell types. The extracellular N-terminal domain of thereceptor is highly glycosylated and binds EGF antibodies thatselectively bind to EGFR. Agents that competitively bind to EGFR havebeen used to treat certain types of cancer, since many tumors ofmesodermal and ectodermal origin overexpress the EGF receptor. Forexample, the EGF receptor has been shown to be overexpressed in manygliomas, squamous cell carcinomas, breast carcinomas, melanomas,invasive bladder carcinomas and esophageal cancers. Attempts to exploitthe EGFR system for anti-tumor therapy have generally involved the useof monoclonal antibodies against the EGFR. In addition, studies withprimary human mammary tumors have shown a correlation between high EGFRexpression and the presence of metastases, higher rates ofproliferation, and shorter individual survival.

Herlyn et al., in U.S. Pat. No. 5,470,571, disclose the use ofradiolabeled Mab 425 for treating gliomas that express EGFR. Herlyn etal. report that anti-EGFR antibodies may either stimulate or inhibitcancer cell growth and proliferation. Other monoclonal antibodies havingspecificity for EGFR, either alone or conjugated to a cytotoxiccompound, have been reported as being effective for treating certaintypes of cancer. Bendig et al, in U.S. Pat. No. 5,558,864, disclosetherapeutic anti-EGFR Mab's for competitively binding to EGFR. Heimbrooket al., in U.S. Pat. No. 5,690,928, disclose the use of EGF fused to aPseudomonas species-derived endotoxin for the treatment of bladdercancer. Brown et al., in U.S. Pat. No. 5,859,018, disclose a method fortreating diseases characterized by cellular hyperproliferation mediatedby, inter alia, EGF.

Chemotherapeutic Modes of Administration

People diagnosed as having cancer are frequently treated with single ormultiple chemotherapeutic agents to kill cancer cells at the primarytumor site or at distant sites to where cancer has metastasized.Chemotherapy treatment is typically given either in a single or inseveral large doses or over variable times of weeks to months. However,repeated or high dose cycles of chemotherapy may be responsible forincreased toxicities and severe side effects.

New studies suggest that metronomic chemotherapy, the low-dose andfrequent administration of cytotoxic agents without prolonged drug-freebreaks, targets activated endothelial cells in the tumor vasculature. Anumber of preclinical studies have demonstrated superior anti-tumorefficacy, potent antiangiogenic effects, and reduced toxicity and sideeffects (e.g., myelosuppression) of metronomic regimes compared tomaximum tolerated dose (MTD) counterparts (Bocci, et al., Cancer Res,62:6938-6943, (2002); Bocci, et al., PNAS, vol, 100(22):12917-12922,(2003); and Bertolini, et al., Cancer Res, 63(15):4342-4346, (2003)). Itremains unclear whether all chemotherapeutic drugs exert similar effectsor whether some are better suited for such regimes than others.Nevertheless, metronomic chemotherapy appears to be effective inovercoming some of the major shortcomings associated with chemotherapy.

Chemotherapeutic Agents

Paclitaxel has been shown to have significant antineoplastic andanticancer effects in drug-refractory ovarian cancer and has shownexcellent antitumor activity in a wide variety of tumor models, and alsoinhibits angiogenesis when used at very low doses (Grant et al., Int. J.Cancer, 2003). The poor aqueous solubility of paclitaxel, however,presents a problem for human administration. Indeed, the delivery ofdrugs that are inherently insoluble or poorly soluble in an aqueousmedium can be seriously impaired if oral delivery is not effective.Accordingly, currently used paclitaxel formulations (e.g., Taxol®)require a Cremophor® to solubilize the drug. The presence of Cremophor®in this formulation has been linked to severe hypersensitivity reactionsin animals (Lorenz et al., Agents Actions 7:63-67 (1987)) and humans(Weiss et al., J. Clin. Oncol. 8:1263-68 (1990)) and consequentlyrequires premedication of individuals with corticosteroids(dexamethasone) and antihistamines. It was also reported that clinicallyrelevant concentrations of the formulation vehicle Cremophor® EL inTaxol® nullify the antiangiogenic activity of paclitaxel, suggestingthat this agent or other anticancer drugs formulated in Cremophor® ELmay need to be used at much higher doses than anticipated to achieveeffective metronomic chemotherapy (Ng et al., Cancer Res., 64:821-824(2004)). As such, the advantage of the lack of undesirable side effectsassociated with low-dose paclitaxel regimes vs. conventional MTDchemotherapy may be compromised. See also U.S. Patent Pub. No.2004/0143004; WO00/64437.

Abraxane® is a Cremophor® EL-Free Nanoparticle Albumin-Bound Paclitaxel

Preclinical models have shown significant improvement in the safety andefficacy of Abraxane® compared with Taxol® (Desai et al.,EORTC-NCI-AACR, 2004) and in individuals with metastatic breast cancer(O'Shaughnessy et al., San Antonio Breast Cancer Symposium, Abstract#1122, December 2003). This is possibly due to the absence ofsurfactants (e.g., Cremophor® or Tween® 80, used in Taxol® andTaxotere®, respectively) in Abraxane®, and/or preferential utilizationof an albumin-based transport mechanism utilizing gp60/caveolae onmicrovascular endothelial cells (Desai et al., EORTC-NCI-AACR, 2004). Inaddition, both Cremophor® and Tween® 80 have been shown to stronglyinhibit the binding of paclitaxel to albumin, possibly affecting albuminbased transport (Desai et al., EORTC-NCI-AACR, 2004).

IDN5109 (Ortataxel) is a new taxane, currently in phase II, selected forits lack of cross-resistance in tumor cell lines expressing themultidrug resistant phenotype (MDR/Pgp) and inhibition of P-glycoprotein(Pgp) (Minderman; Cancer Chemother. Pharmacol. 2004; 53:363-9). Due toits hydrophobicity, IDN5109 is currently formulated in the surfactantTween® 80 (same vehicle as Taxotere®). Removal of surfactants fromtaxane formulations e.g., in the case of nanoparticle albumin-boundpaclitaxel (Abraxane®) showed improvements in safety and efficacy overtheir surfactant containing counterparts (O'Shaughnessy et al., SanAntonio Breast Cancer Symposium, Abstract #1122, December 2003). Tween®80 also strongly inhibited the binding of the taxane, paclitaxel, toalbumin, possibly compromising albumin based drug transport via the gp60receptor on microvessel endothelial cells (Desai et al., EORTC-NCI-AACR,2004).

The antitumor activity of colchicine, which is the major alkaloid of theautumn crocus, Colchicum autumnale, and the African climbing lily,Gloriosa superba, was first reported at the beginning of the 20^(th)century. The elucidation of its structure was finally completed fromX-ray studies and a number of total syntheses (see Shiau et al., J.Pharm. Sci. 1978, 67(3) 394-397). Colchicine is thought to be a mitoticpoison, particularly in tyhmic, intestinal, and hermatopoietic cells,which acts as a spindle poison and blocks the kinesis. Its effect on themitotic spindle is thought to represent a special case of its effects onvarious organized, labile, fibrillar systems concerned with structureand movement.

Thiocolchicine dimer IDN5404 was selected for its activity in humanovarian subline resistant to cisplatin and topotecan A2780-CIS andA2780-TOP. This effect was related to dual mechanisms of action, i.e.,microtubule activity as in Vinca alkaloids and a topoisomerase Iinhibitory effect different from camptothecin. (Raspaglio, BiochemicalPharmacology 69:113-121 (2005)).

It has been found that nanoparticle compositions of a taxane (such asalbumin bound paclitaxel (Abraxane®)) have significantly lowertoxicities than other taxanes like Taxol® and Taxotere® withsignificantly improved outcomes in both safety and efficacy.

Combination chemotherapy, e.g., combining one or more chemotherapeuticagents or other modes of treatment, e.g., combining for example,chemotherapy with radiation or surgery and chemotherapy, have been foundto be more successful than single agent chemotherapeutics or individualmodes of treatment respectively.

Other references include U.S. Pub. No. 2006/0013819; U.S. Pub. No.2006/0003931; WO05/117986; WO05/117978; and WO05/000900.

More effective treatments for proliferative diseases, especially cancer,are needed.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entirety. In countries where this isappropriate, this application incorporates by reference any applicationto which this application claims the priority benefit.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for the treatment ofproliferative diseases such as cancer. The invention providescombination therapy methods of treating proliferative diseases (such ascancer), comprising a) a first therapy comprising administering to anindividual an effective amount of a composition comprising nanoparticlescomprising a taxane (such as paclitaxel) and a carrier protein (such asalbumin) and b) a second therapy, such as chemotherapy, radiationtherapy, surgery, or combinations thereof. In another aspect, there areprovided methods of administering to an individual a compositioncomprising nanoparticles comprising a taxane (such as paclitaxel) and acarrier protein (such as albumin) based on a metronomic dosing regime.

In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), and b) an effective amount of at least one otherchemotherapeutic agent. In some variations, the invention provides amethod of treating a proliferative disease (such as cancer) in anindividual comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising paclitaxeland an albumin (such as Abraxane®), and b) an effective amount of atleast one other chemotherapeutic agent. In some variations, thechemotherapeutic agent is any of (and in some variations selected fromthe group consisting of) antimetabolites (including nucleoside analogs),platinum-based agents, alkylating agents, tyrosine kinase inhibitors,anthracycline antibiotics, vinca alkloids, proteasome inhibitors,macrolides, and topoisomerase inhibitors. In some variations, thechemotherapeutic agent is a platinum-based agent, such as carboplatin.

In some variations, there is provided a method of treating aproliferative disease (such as cancer, for example breast cancer) in anindividual, comprising administering to the individual: a) an effectiveamount of a composition comprising nanoparticles comprising taxane (suchas paclitaxel) and a carrier protein, and b) an effective amount of ananti-VEGF antibody (such as bevacizumab, for example Avastin®). In somevariations, there is provided a method of inhibiting tumor metastasis(such as breast cancer metastasis) in an individual, comprisingadministering to the individual: a) an effective amount of a compositioncomprising nanoparticles comprising taxane (such as paclitaxel) and acarrier protein, and b) an effective amount of an anti-VEGF antibody(such as bevacizumab, for example Avastin®). In some variations, theeffective amounts of the nanoparticle composition and the anti-VEGFantibody synergistically inhibit cell proliferation or metastasis.

In some variations, the effective amount of taxane in the nanoparticlecomposition is between about 45 mg/m² to about 350 mg/m² and theeffective amount of anti-VEGF antibody is greater than 1 mg/kg to lessthan 10 mg/kg or greater than 15 mg/kg to less than 20 mg/kg. In somevariations, the effective amount of taxane in the nanoparticlecomposition is between about 80 mg/m² to about 150 mg/m² and theeffective amount of anti-VEGF antibody is greater than 1 mg/kg to lessthan 10 mg/kg or greater than 15 mg/kg to less than 20 mg/kg. In somevariations, the effective amount of taxane (e.g., paclitaxel) in thenanoparticle composition is about 100 mg/m². In some variations, thetaxane in the nanoparticle composition is administered weekly. In somevariations, the effective amount of taxane in the nanoparticlecomposition is between about 170 mg/m² to about 200 mg/m² and theeffective amount of anti-VEGF antibody is greater than 1 mg/kg to lessthan 10 mg/kg or greater than 15 mg/kg to less than 20 mg/kg. In somevariations, the taxane in a nanoparticle composition is administeredevery two weeks. In some variations, the effective amount of taxane inthe nanoparticle composition is between about 200 mg/m² to about 350mg/m² and the effective amount of anti-VEGF antibody is greater than 1mg/kg to less than 10 mg/kg or greater than 15 mg/kg to less than 20mg/kg. In some variations, the effective amount of taxane (e.g.,paclitaxel) in the nanoparticle composition is about 260 mg/m². In somevariations, the taxane in the nanoparticle composition is administeredevery three weeks. In some variations of any of the above methods, theeffective amount of anti-VEGF antibody is about 2 mg/kg, about 4 mg/kg,about 6 mg/kg, or about 8 mg/kg. In some variations, the anti-VEGFantibody is administered every two weeks or every three weeks. In somevariations of the above dosages and/or administrations, the taxane ispaclitaxel. In some variations of the above dosages and/oradministrations, the anti-VEGF antibody is bevacizumab (Avastin®). Insome variations of the above dosages and/or administrations, the taxaneis paclitaxel and the anti-VEGF antibody is bevacizumab (Avastin®)

In some variations, there is provided a method of treating aproliferative disease (such as cancer, for example breast cancer) in anindividual, comprising administering to the individual: a) an effectiveamount of a composition comprising taxane, and b) an effective amount ofan anti-VEGF antibody (such as bevacizumab, for example Avastin®),wherein the effective amount of the anti-VEGF antibody is an amounteffective to suppress taxane-mediated induction of VEGF in vivo. In somevariations, there is provided a method of inhibiting tumor metastasis(such as breast cancer metastasis) in an individual, comprisingadministering to the individual: a) an effective amount of a compositioncomprising taxane, and b) an effective amount of an anti-VEGF antibody(such as bevacizumab, for example Avastin®), wherein the effectiveamount of the anti-VEGF antibody is an amount effective to suppresstaxane-mediated induction of VEGF in vivo. In some variations, thetaxane-mediated induction of VEGF in vivo is taxane-mediated inductionof VEGF-A. In some variations, the taxane is paclitaxel. In somevariations, the taxane is docetaxel. In some variations, the compositioncomprising a taxane is a composition comprising nanoparticles comprisinga taxane and a carrier protein. In some variations, the nanoparticlescomprising a taxane are nanoparticles comprising paclitaxel. In somevariations, the nanoparticles comprising a taxane are nanoparticlescomprising docetaxel.

In some variations, the effective amount of taxane in the composition isbetween about 45 mg/m² to about 350 mg/m² and the effective amount ofanti-VEGF antibody is between about 1 mg/kg to about 20 mg/kg. In somevariations, the effective amount of taxane in the composition is betweenabout 80 mg/m² to about 150 mg/m² and the effective amount of anti-VEGFantibody is between about 1 mg/kg to about 20 mg/kg. In some variations,the effective amount of taxane (e.g., paclitaxel) in the composition isabout 100 mg/m². In some variations, the taxane is administered weekly.In some variations, the effective amount of taxane in the composition isbetween about 170 mg/m² to about 200 mg/m² and the effective amount ofanti-VEGF antibody is between about 1 mg/kg to about 20 mg/kg. In somevariations, the effective amount of taxane in the composition is betweenabout 200 mg/m² to about 350 mg/m² and the effective amount of anti-VEGFantibody is between about 1 mg/kg to about 20 mg/kg. In some variations,the taxane is administered every two weeks. In some variations, theeffective amount of taxane (e.g., paclitaxel) in the composition isabout 260 mg/m². In some variations, the taxane is administered everythree weeks. In some variations of any of the above methods, theeffective amount of anti-VEGF antibody is greater than 1 mg/kg to lessthan 10 mg/kg or greater than 15 mg/kg to less than 20 mg/kg. In somevariations, the effective amount of anti-VEGF antibody is between about5 to about 10 mg/kg. In some variations of any of the above methods, theeffective amount of anti-VEGF antibody is about 2 mg/kg, about 4 mg/kg,about 6 mg/kg, about 8 mg/kg, about 10 mg/kg, about 12 mg/kg, or about15 mg/kg. In some variations, the anti-VEGF antibody is administeredevery two weeks or every three weeks. In some variations of the abovedosages and/or administrations, the taxane is paclitaxel. In somevariations of the above dosages and/or administrations, the anti-VEGFantibody is bevacizumab (Avastin®). In some variations of the abovedosages and/or administrations, the taxane is paclitaxel and theanti-VEGF antibody is bevacizumab (Avastin®)

In some variations, the composition comprising nanoparticles (alsoreferred to as “nanoparticle composition”) and the chemotherapeuticagent are administered simultaneously, either in the same composition orin separate compositions. In some variations, the nanoparticlecomposition and the chemotherapeutic agent are administeredsequentially, i.e., the nanoparticle composition is administered eitherprior to or after the administration of the chemotherapeutic agent. Insome variations, the administration of the nanoparticle composition andthe chemotherapeutic agent are concurrent, i.e., the administrationperiod of the nanoparticle composition and that of the chemotherapeuticagent overlap with each other. In some variations, the administration ofthe nanoparticle composition and the chemotherapeutic agent arenon-concurrent. For example, in some variations, the administration ofthe nanoparticle composition is terminated before the chemotherapeuticagent is administered. In some variations, the administration of thechemotherapeutic agent is terminated before the nanoparticle compositionis administered.

In some variations, the first therapy taxane is nano-particle albuminbound paxlitaxel, described, for example, in U.S. Pat. No. 6,566,405,and commercially available under the tradename Abraxane®. In addition,the first therapy taxane is also considered to be nanoparticle albuminbound docetaxel described for example in U.S. Patent ApplicationPublication 2005/0004002A1. In some variations, the anti-VEGF antibodyis bevacizumab (i.e., Avastin®).

In another aspect, there is provided a method of treating aproliferative disease (such as cancer) in an individual comprising a) afirst therapy comprising administering to the individual a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), and b) a second therapy comprising radiation therapy,surgery, or combinations thereof. In some variations, there is provideda method of treating a proliferative disease (such as cancer) in anindividual comprising a) a first therapy comprising administering to theindividual a composition comprising nanoparticles comprising paclitaxeland an albumin (such as Abraxane®), and b) a second therapy comprisingradiation therapy, surgery, or combinations thereof. In some variations,the second therapy is radiation therapy. In some variations, the secondtherapy is surgery. In some variations, the first therapy is carried outprior to the second therapy. In some variations, the first therapy iscarried out after the second therapy.

In another aspect, the method comprises administering to a mammal havinga proliferative disease (such as cancer) a combination therapycomprising a first therapy comprising a taxane and a second therapyselected from the group consisting of chemotherapeutic agent andradiation or combinations thereof. The combination therapy may beadministered in any of a variety of ways such as sequentially orsimultaneously, and if sequential, the taxane may be administered beforeor after the second therapy although it is preferred that the firsttherapy comprising a taxane is administered first. It will also beunderstood that the second therapy can include more than onechemotherapeutic agent.

The present invention also provides metronomic therapy regimes. In somevariations, there is provided a method of administering a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), wherein the nanoparticle composition is administered over aperiod of at least one month, wherein the interval between eachadministration is no more than about a week, and wherein the dose oftaxane at each administration is about 0.25% to about 25% of its maximumtolerated dose following a traditional dosing regime. In somevariations, there is provided a method of administering a compositioncomprising nanoparticles comprising paclitaxel and an albumin (such asAbraxane®), wherein the nanoparticle composition is administered over aperiod of at least one month, wherein the interval between eachadministration is no more than about a week, and wherein the dose ofpaclitaxel at each administration is about 0.25% to about 25% of itsmaximum tolerated dose following a traditional dosing regime. In somevariations, the dose of the taxane (such as paclitaxel, for exampleAbraxane®) per administration is less than about any of 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 18%, 20%, 22%, 24%, or25% of the maximum tolerated dose. In some variations, the nanoparticlecomposition is administered at least about any of 1×, 2×, 3×, 4×, 5×,6×, 7× (i.e., daily) a week. In some variations, the intervals betweeneach administration are less than about any of 7 days, 6 days, 5 days, 4days, 3 days, 2 days, and 1 day. In some variations, the nanoparticlecomposition is administered over a period of at least about any of 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30 and 36 months.

In some variations, there is provided a method of administering acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), wherein the taxane is administered over aperiod of at least one month, wherein the interval between eachadministration is no more than about a week, and wherein the dose of thetaxane at each administration is about 0.25 mg/m² to about 25 mg/m². Insome variations, there is provided a method of administering acomposition comprising nanoparticles comprising paclitaxel and analbumin (such as Abraxane®) and a carrier protein (such as albumin),wherein the paclitaxel is administered over a period of at least onemonth, wherein the interval between each administration is no more thanabout a week, and wherein the dose of the taxane at each administrationis about 0.25 mg/m² to about 25 mg/m². In some variations, the dose ofthe taxane (such as paclitaxel, for example Abraxane®) peradministration is less than about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 18, 20, 22, and 25 mg/m². In some variations, thenanoparticle composition is administered at least about any of 1×, 2×,3×, 4×, 5×, 6×, 7× (i.e., daily) a week. In some variations, theintervals between each administration are less than about any of 7 days,6 days, 5 days, 4 days, 3 days, 2 days, and 1 day. In some variations,the nanoparticle composition is administered over a period of at leastabout any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30 and 36months.

The methods of the invention generally comprise administration of acomposition comprising nanoparticles comprising a taxane and a carrierprotein. In some variations, the nanoparticle composition comprisesnanoparticles comprising paclitaxel and an albumin. In some variations,the paclitaxel/albumin nanoparticles have an average diameter of nogreater than about 200 nm. In some variations, the paclitaxel/albuminnanoparticle composition is substantially free (such as free) ofsurfactant (such as Cremophor). In some variations, the weight ratio ofthe albumin to paclitaxel in the composition is about 18:1 or less, suchas about 9:1 or less. In some variations, the paclitaxel is coated withalbumin. In some variations, the paclitaxel/albumin nanoparticles havean average diameter of no greater than about 200 nm and thepaclitaxel/albumin composition is substantially free (such as free) ofsurfactant (such as Cremophor). In some variations, thepaclitaxel/albumin nanoparticles have an average diameter of no greaterthan about 200 nm and the paclitaxel is coated with albumin. Othercombinations of the above characteristics are also contemplated. In somevariations, the nanoparticle composition is Abraxane®. Nanoparticlecompositions comprising other taxanes (such as docetaxel and ortataxel)may also comprise one or more of the above characteristics.

In some variations, there is provided a method of treating aproliferative disease in an individual, comprising administering to theindividual: a) an effective amount of a composition comprisingnanoparticles comprising taxane and a carrier protein, and b) aneffective amount of an anti-VEGF antibody, wherein the effective amountof taxane in the nanoparticle composition is between about 45 mg/m² toabout 350 mg/m² and the effective amount of anti-VEGF antibody isgreater than 1 mg/kg to less than 10 mg/kg or greater than 15 mg/kg toless than 20 mg/kg. In some variations, there is provided a method oftreating a proliferative disease in an individual comprisingadministering to the individual: (1) an effective amount of acomposition comprising a taxane, and (2) an effective amount of ananti-VEGF antibody, wherein the effective amount of the anti-VEGFantibody is an amount effective to suppress taxane-mediated induction ofVEGF in vivo. In some variations, the composition comprising the taxaneis a composition comprising nanoparticles comprising a taxane and acarrier protein.

In some variations, the proliferative disease is cancer. In somevariations, the cancer is breast cancer. In some variations, theanti-VEGF antibody is bevacizumab. In some variations, the effectiveamount of the anti-VEGF antibody is about 6 mg/kg. In some variations,the effective amount of the anti-VEGF antibody is about 8 mg/kg. In somevariations, the effective amount of taxane in the nanoparticlecomposition is between about 80 mg/m² to about 150 mg/m² of taxane inthe nanoparticle composition. In some variations, the effective amountof taxane in the nanoparticle composition is between about 200 mg/m² toabout 350 mg/m² of taxane in the nanoparticle composition. In somevariations, the nanoparticle composition and the anti-VEGF antibody areadministered sequentially to the individual. In some variations, thenanoparticle composition is administered for at least one cycles priorto the administration of the anti-VEGF antibody. In some variations, theadministration of the nanoparticle composition is followed by theadministration of an anti-VEGF antibody for at least about 3 weeks. Insome variations, the method comprises administration taxane in ananoparticle composition concurrent with administration of anti-VEGFantibody. In some variations, the taxane is paclitaxel. In somevariations, the average diameter of the nanoparticles in the compositionis no greater than about 200 nm. In some variations, the carrier proteinis albumin. In some variations, the weight ratio of the albumin and thetaxane in the nanoparticle composition is less than about 9:1. In somevariations, the nanoparticle composition is free of Cremophor. In somevariations, the individual is human.

In some variations, there is provided a method of inhibiting tumormetastasis in an individual, comprising administering to the individual:a) an effective amount of a composition comprising nanoparticlescomprising taxane and a carrier protein, and b) an effective amount ofan anti-VEGF antibody, wherein the effective amount of taxane in thenanoparticle composition is between about 45 mg/m² to about 350 mg/m²and the effective amount of anti-VEGF antibody is greater than 1 mg/kgto less than 10 mg/kg or greater than 15 mg/kg to less than 20 mg/kg. Insome variations, there is provided a method of inhibiting tumormetastasis in an individual comprising administering to the individual:(1) an effective amount of a composition comprising a taxane, and (2) aneffective amount of an anti-VEGF antibody, wherein the effective amountof the anti-VEGF antibody is an amount effective to suppresstaxane-mediated induction of VEGF in vivo. In some variations, thecomposition comprising the taxane is a composition comprisingnanoparticles comprising a taxane and a carrier protein.

In some variations, the tumor metastasis is metastasis to lymph node. Insome variations, the tumor metastasis is metastasis to the lung. In somevariations, the tumor metastasis is metastasis of breast cancer. In somevariations, the anti-VEGF antibody is bevacizumab. In some variations,the effective amount of the anti-VEGF antibody is about 6 mg/kg. In somevariations, the effective amount of the anti-VEGF antibody is about 8mg/kg. In some variations, the effective amount of taxane in thenanoparticle composition is between about 80 mg/mg/m² to about 150 mg/m²of taxane in the nanoparticle composition. In some variations, theeffective amount of taxane in the nanoparticle composition is betweenabout 200 mg/m² to about 350 mg/m² of taxane in the nanoparticlecomposition. In some variations, the nanoparticle composition and theanti-VEGF antibody are administered sequentially to the individual. Insome variations, the nanoparticle composition is administered for atleast one cycles prior to the administration of the anti-VEGF antibody.In some variations, the administration of the nanoparticle compositionis followed by the administration of an anti-VEGF antibody for at leastabout 3 weeks. In some variations, the method comprises administrationof taxane in a nanoparticle composition concurrent with administrationof anti-VEGF antibody. In some variations, the taxane is paclitaxel. Insome variations, the average diameter of the nanoparticles in thecomposition is no greater than about 200 nm. In some variations, thecarrier protein is albumin. In some variations, the weight ratio of thealbumin and the taxane in the nanoparticle composition is less thanabout 9:1. In some variations, the nanoparticle composition is free ofCremophor. In some variations, the individual is human. In somevariations, at least about 40% of metastasis is inhibited. In somevariations, at least about 80% of metastasis is inhibited.

These and other aspects and advantages of the present invention willbecome apparent from the subsequent detailed description and theappended claims. It is to be understood that one, some, or all of theproperties of the various variations described herein may be combined toform other variations of the present invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A shows the effect of ABI-007 on rat aortic ring angiogenesis.FIG. 1B shows the effect of ABI-007 on human endothelial cellproliferation. FIG. 1C shows the effect of ABI-007 on endothelial celltube formation.

FIG. 2 shows the determination of an optimal biological dose of ABI-007for metronomic dosing. Shown are the levels of viable circulatingendothelial progenitors (CEPs) in peripheral blood of Balb/cJ mice inresponse to escalating doses of ABI-007. Untr'd, untreated control; S/A,saline/albumin vehicle control. Bars, mean±SE. * Significantly (p<0.05)different from the untreated control.

FIGS. 3A and 3B show the effects of ABI-007 and Taxol® used inmetronomic or MTD regimes on MDA-MB-231 (A) and PC3 (B) tumor growthtumor-bearing SCID mice. FIGS. 3C and 3D show the effects of ABI-007 andTaxol® used in metronomic or MTD regimes on the body weight ofMDA-MB-231 (C) and PC3 (D) tumor-bearing SCID mice.

FIGS. 4A and 4B show changes in the levels of viable circulatingendothelial progenitors (CEPs) in peripheral blood of MDA-MB-231 (FIG.4A) and PC3 (FIG. 4B) tumor-bearing SCID mice after treatment with A,saline/albumin; B, Cremophor EL control; C, metronomic Taxol® 1.3 mg/kg;D, E, and F, metronomic ABI-007 3, 6, and 10 mg/kg, respectively; G, MTDTaxol®; H, MTD ABI-007. Bars, mean±SE.^(a) Significantly (p<0.05)different from saline/albumin vehicle control.^(b) Significantly(p<0.05) different from Cremophor EL vehicle control.

FIG. 5A shows intratumoral microvessel density of MDA-MB-231 (▪) and PC3(□) xenografts treated with A, saline/albumin; B, Cremophor EL control;C, metronomic Taxol® 1.3 mg/kg; D, E, and F, metronomic ABI-007 3, 6,and 10 mg/kg, respectively; G, MTD Taxol; H, MTD ABI-007. Bars, mean±SE.FIGS. 5B and 5C show the correlation between intratumoral microvesseldensity and the number of viable CEPs in peripheral blood in MDA-MB-231(FIG. 5B) and PC3 (FIG. 5C) tumor-bearing SCID mice.

FIG. 6 shows the effects of ABI-007 or Taxol used in metronomic or MTDregimes on basic fibroblast growth factor (bFGF)-induced angiogenesis inmatrigel plugs injected subcutaneously into the flanks of Balb/cJ mice.Treatments-A, saline/albumin; B, Cremophor EL control; C, metronomicTaxol 1.3 mg/kg; D, E, and F, metronomic ABI-007 3, 6, and 10 mg/kg,respectively; G, MTD Taxol; H, MTD ABI-007. Matrigel implanted withoutbFGF (-bFGF) served as negative control. Bars, mean±SE.

FIG. 7A and FIG. 7B show the cytotoxic activity of nab-rapamycin incombination with Abraxane® on vascular smooth muscle cells. Cytotoxicitywas evaluated by staining with ethidium homodimer-1 (FIG. 7A) or bystaining with calcein (FIG. 7B).

FIG. 8 shows the cytotoxic activity of nab-rapamycin in combination withAbraxane® in a HT29 human colon carcinoma xenograft model.

FIG. 9 shows the cytotoxic activity of nab-17-AAG in combination withAbraxane® in a H358 human lung carcinoma xenograft model.

FIGS. 10A and 10B show necrosis in MDA-MB-231 tumor cells aftertreatment with saline control or Abraxane®. FIGS. 10C and 10D showhypoxia in MDA-MB-231 cells after treatment with saline control orAbraxane®. Arrows indicate sites of necrosis (10A and 10B) or sites ofhypoxia (10C and 10D).

FIGS. 11A and 11B show the effect of VEGF-A and Avastin® onAbraxane®-treated cells in cytotoxicity and clonogenic assays. In FIG.11A, results are shown as viable cells as a percentage of untreatedcells. Dark circles indicate cells treated with Abraxane® alone; opencircles indicate cells treated with Abraxane® and VEGF-A; dark trianglesindicate cells treated with Abraxane® and Avastin®. In FIG. 11B, resultsare shown as the mean number of cell colonies per plate.

FIG. 12 shows the effect of Abraxane® and Avastin® treatment on thegrowth of MDA-MB-231 breast tumor xenografts. Dark squares indicate meantumor volume in saline-treated mice; dark circles indicate mean tumorvolume in Abraxane®-treated mice; dark diamonds indicate mean tumorvolume in Avastin®-treated mice; open diamonds indicate mean tumorvolume in Abraxane®+Avastin® (2 mg/kg)-treated mice; open circlesindicate mean tumor volume in Abraxane®+Avastin® (4 mg/kg)-treated mice;triangles indicate mean tumor volume in Abraxane®+Avastin® (8mg/kg)-treated mice. Two bars labeled ABX indicate the two Abraxane®treatment cycles.

FIGS. 13A and 13B show the effect of Abraxane® and Avastin® treatment onmetastasis of luciferase-expressing MDA-MB-231 tumor cells to the lymphnodes and lungs in tumor-bearing mice. Results are shown as levels ofluciferase activity in lymph node or lung cellular extracts.

FIG. 14 shows the effect of solvent-based (i.e., Taxol®) andsolvent-free (i.e., nab-paclitaxel, Abraxane®) formulations ofpaclitaxel on tumor volume (FIG. 14A) and reactionary angiogenesis (FIG.14B) in paclitaxel-sensitive (MX-1 and MES-SA) and paclitaxel-resistant(MES-SA/Dx5) xenografts.

FIG. 15 shows that administration of Avastin® in combination withAbraxane® to mice bearing MDA-MS-231 human breast cancer xenograftssignificantly improves tumor suppression induced by Abraxane® alone.

FIG. 16 shows that combination therapy with Abraxane® and Avastin® butnot Abraxane® or Avastin® alone resulted in sustainable tumorregressions in all treated mice for at least 95 days after tumorimplantation.

FIG. 17 shows that combination therapy with Abraxane® and Avastin® butnot Abraxane® or Avastin® alone resulted in inhibition of lymphatic(FIG. 17A) and pulmonary (FIG. 17B) metastasis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of combination therapy comprisinga first therapy comprising administration of nanoparticles comprising ataxane and a carrier protein (such as albumin) in conjunction with asecond therapy such as radiation, surgery, administration of at leastone other chemotherapeutic agent, or combinations thereof. The inventionalso provides methods of metronomic therapy.

The present invention involves the discovery that Abraxane®, due to itssuperior anti-tumor activity and reduced toxicity and side effects, canbe administered in combination with other therapeutic drugs and/ortreatment modalities and can also be used in metronomic chemotherapy.Due to significantly improved safety profiles with compositionscomprising drug/carrier protein nanoparticles (such as Abraxane®), webelieve that combination chemotherapy with such nanoparticlecompositions (such as Abraxane®) is more effective than combinationchemotherapy with other drugs. In addition the use of nanoparticlecomposition (such as Abraxane®) in combination with radiation is alsobelieved to be more effective than combination of other agents withradiation. Thus, the nanoparticle compositions (especially apaclitaxel/albumin nanoparticle composition, such as Abraxane®), whenused in combination with other chemotherapeutic agents or when combinedwith other treatment modalities, should be very effective and overcomethe deficiencies of surgery, radiation treatment, and chemotherapy inthe treatment of proliferative disease (such as cancer).

The present invention in one its variations is the use of a firsttherapy comprising a taxane, such as Abraxane®, in combination with asecond therapy such as another chemotherapeutic agent or agents,radiation, or the like for treating proliferative diseases such ascancer. The first therapy comprising a taxane and second therapy can beadministered to a mammal having the proliferative sequentially, or theycan be co-administered, and even administered simultaneously in the samepharmaceutical composition.

Further, a metronomic dosing regime using Abraxane® has been found to bemore effective than the traditional MTD dosing schedule of the same drugcomposition. Such metronomic dosing regime of Abraxane® has also beenfound to be more effective than metronomic dosing of Taxol®.

The methods described herein are generally useful for treatment ofdiseases, particularly proliferative diseases. As used herein,“treatment” is an approach for obtaining beneficial or desired clinicalresults. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, any one or more of: alleviationof one or more symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, preventing or delaying spread(e.g., metastasis) of disease, preventing or delaying occurrence orrecurrence of disease, delay or slowing of disease progression,amelioration of the disease state, and remission (whether partial ortotal). Also encompassed by “treatment” is a reduction of pathologicalconsequence of a proliferative disease. The methods of the inventioncontemplate any one or more of these aspects of treatment.

As used herein, a “proliferative disease” is defined as a tumor disease(including benign or cancerous) and/or any metastases, wherever thetumor or the metastasis are located, more especially a tumor selectedfrom the group comprising one or more of (and in some variationsselected from the group consisting of) breast cancer, genitourinarycancer, lung cancer, gastrointestinal cancer, epidermoid cancer,melanoma, ovarian cancer, pancreatic cancer, neuroblastoma, colorectalcancer, head and neck cancer. In a broader sense of the invention, aproliferative disease may furthermore be selected fromhyperproliferative conditions such as hyperplasias, fibrosis (especiallypulmonary, but also other types of fibrosis, such as renal fibrosis),angiogenesis, psoriasis, atherosclerosis and smooth muscle proliferationin the blood vessels, such as stenosis or restenosis followingangioplasty. In some variations, the proliferative disease is cancer. Insome variations, the proliferative disease is a non-cancerous disease.In some variations, the proliferative disease is a benign or malignanttumor. Where hereinbefore and subsequently a tumor, a tumor disease, acarcinoma or a cancer are mentioned, also metastasis in the originalorgan or tissue and/or in any other location are implied alternativelyor in addition, whatever the location of the tumor and/or metastasis is.

The term “effective amount” used herein refers to an amount of acompound or composition sufficient to treat a specified disorder,condition or disease such as ameliorate, palliate, lessen, and/or delayone or more of its symptoms. In reference to cancers or other unwantedcell proliferation, an effective amount comprises an amount sufficientto cause a tumor to shrink and/or to decrease the growth rate of thetumor (such as to suppress tumor growth) or to prevent or delay otherunwanted cell proliferation. In some variations, an effective amount isan amount sufficient to delay development. In some variations, aneffective amount is an amount sufficient to prevent or delay occurrenceand/or recurrence. An effective amount can be administered in one ormore administrations. In the case of cancer, the effective amount of thedrug or composition may: (i) reduce the number of cancer cells; (ii)reduce tumor size; (iii) inhibit, retard, slow to some extent andpreferably stop cancer cell infiltration into peripheral organs; (iv)inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrenceand/or recurrence of tumor; and/or (vii) relieve to some extent one ormore of the symptoms associated with the cancer.

In some variations, there is provided a method of treating a primarytumor. In some variations, there is provided a method of treatingmetastatic cancer (that is, cancer that has metastasized from theprimary tumor). In some variations, there is provided a method oftreating cancer at advanced stage(s). In some variations, there isprovided a method of treating breast cancer (which may be HER2 positiveor HER2 negative), including, for example, advanced breast cancer, stageIV breast cancer, locally advanced breast cancer, and metastatic breastcancer. In some variations, there is provided a method of treating lungcancer, including, for example, non-small cell lung cancer (NSCLC, suchas advanced NSCLC), small cell lung cancer (SCLC, such as advancedSCLC), and advanced solid tumor malignancy in the lung. In somevariations, there is provided a method of treating any of ovariancancer, head and neck cancer, gastric malignancies, melanoma (includingmetastatic melanoma), colorectal cancer, pancreatic cancer, and solidtumors (such as advanced solid tumors). In some variations, there isprovided a method of reducing cell proliferation and/or cell migration.In some variations, there is provided a method of treating any of thefollowing diseases: restenosis, stenosis, fibrosis, angiogenesis,psoriasis, atherosclerosis, and proliferation of smooth muscle cells.The present invention also provides methods of delaying development ofany of the proliferative diseases described herein.

The term “individual” is a mammal, including humans. An individualincludes, but is not limited to, human, bovine, horse, feline, canine,rodent, or primate. In some variations, the individual is human. Theindividual (such as human) may have advanced disease or lesser extent ofdisease, such as low tumor burden. In some variations, the individual isat an early stage of a proliferative disease (such as cancer). In somevariations, the individual is at an advanced stage of a proliferativedisease (such as an advanced cancer). In some variations, the individualis HER2 positive. In some variations, the individual is HER2 negative.

The methods may be practiced in an adjuvant setting. “Adjuvant setting”refers to a clinical setting in which an individual has had a history ofa proliferative disease, particularly cancer, and generally (but notnecessarily) been responsive to therapy, which includes, but is notlimited to, surgery (such as surgical resection), radiotherapy, andchemotherapy. However, because of their history of the proliferativedisease (such as cancer), these individuals are considered at risk ofdevelopment of the disease. Treatment or administration in the “adjuvantsetting” refers to a subsequent mode of treatment. The degree of risk(i.e., when an individual in the adjuvant setting is considered as “highrisk” or “low risk”) depends upon several factors, most usually theextent of disease when first treated. The methods provided herein mayalso be practiced in a neoadjuvant setting, i.e., the method may becarried out before the primary/definitive therapy. In some variations,the individual has previously been treated. In some variations, theindividual has not previously been treated. In some variations, thetreatment is a first line therapy.

It is understood that aspect and variations of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand variations.

As is understood by one skilled in the art, reference to “about” a valueor parameter herein includes (and describes) embodiments that aredirected to that value or parameter per se. For example, descriptionreferring to “about X” includes description of “X”.

Combination Therapy with Chemotherapeutic Agent

The present invention provides methods of treating a proliferativedisease (such as cancer) in an individual, comprising administering tothe individual: a) an effective amount of a composition comprisingnanoparticles comprising a taxane and a carrier protein (such asalbumin); and b) an effective amount of at least one otherchemotherapeutic agent. In some variations, the taxane is any of (and income variations consisting essentially of) paclitaxel, docetaxel, andortataxel. In some variations, the nanoparticle composition comprisesAbraxane®. In some variations, the chemotherapeutic agent is any of (andin some variations selected from the group consisting of) antimetaboliteagents (including nucleoside analogs), platinum-based agents, alkylatingagents, tyrosine kinase inhibitors, anthracycline antibiotics, vincaalkloids, proteasome inhibitors, macrolides, and topoisomeraseinhibitors.

In some variations, the method comprises administering to theindividual: a) an effective amount of a composition comprisingnanoparticles comprising paclitaxel and an albumin; and b) an effectiveamount of at least one other chemotherapeutic agent. In some variations,the paclitaxel/albumin nanoparticles have an average diameter of nogreater than about 200 nm. In some variations, the paclitaxel/albuminnanoparticle composition is substantially free (such as free) ofsurfactant (such as Cremophor). In some variations, the weight ratio ofthe albumin to paclitaxel in the composition is about 18:1 or less, suchas about 9:1 or less. In some variations, the paclitaxel is coated withalbumin. In some variations, the paclitaxel/albumin nanoparticles havean average diameter of no greater than about 200 nm and thepaclitaxel/albumin composition is substantially free (such as free) ofsurfactant (such as Cremophor). In some variations, thepaclitaxel/albumin nanoparticles have an average diameter of no greaterthan about 200 nm and the paclitaxel is coated with albumin. In somevariations, the nanoparticle composition is Abraxane®

In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual comprisingadministering to the individual a) an effective amount of Abraxane®, andb) an effective amount of at least one other chemotherapeutic agent.Preferred drug combinations for sequential or co-administration orsimultaneous administration with Abraxane® are those which show enhancedantiproliferative activity when compared with the single componentsalone, especially combinations that that lead to regression ofproliferative tissues and/or cure from proliferative diseases.

The chemotherapeutic agents described herein can be the agentsthemselves, pharmaceutically acceptable salts thereof, andpharmaceutically acceptable esters thereof, as well as steroisomers,enantiomers, racemic mixtures, and the like. The chemotherapeutic agentor agents as described can be administered as well as a pharmaceuticalcomposition containing the agent(s), wherein the pharmaceuticalcomposition comprises a pharmaceutically acceptable carrier vehicle, orthe like.

The chemotherapeutic agent may be present in a nanoparticle composition.For example, in some variations, there is provided a method of treatinga proliferative disease (such as cancer) in an individual, comprisingadministering to the individual: a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin); and b) an effective amount of a composition comprisingnanoparticles comprising at least one other chemotherapeutic agent and acarrier protein (such as albumin). In some variations, the methodcomprises administering to the individual: a) an effective amount of acomposition comprising nanoparticles comprising paclitaxel and analbumin (such as Abraxane®); and b) an effective amount of a compositioncomprising nanoparticles comprising at least one other chemotherapeuticagent and a carrier protein (such as albumin). In some variations, thechemotherapeutic agent is any of (and in some variations selected fromthe group consisting of) thiocolchicine or its derivatives (such asdimeric thiocolchicine, including for example nab-5404, nab-5800, andnab-5801), rapamycin or its derivatives, and geldanamycin or itsderivatives (such as 17-allyl amino geldanamycin (17-AAG)). In somevariations, the chemotherapeutic agent is rapamycin. In some variations,the chemotherapeutic agent is 17-AAG.

An exemplary and non-limiting list of chemotherapeutic agentscontemplated is provided herein. Suitable chemotherapeutic agentsinclude, for example, vinca alkaloids, agents that disrupt microtubuleformation (such as colchicines and its derivatives), anti-angiogenicagents, therapeutic antibodies, EGFR targeting agents, tyrosine kinasetargeting agent (such as tyrosine kinase inhibitors), transitional metalcomplexes, proteasome inhibitors, antimetabolites (such as nucleosideanalogs), alkylating agents, platinum-based agents, anthracyclineantibiotics, topoisomerase inhibitors, macrolides, therapeuticantibodies, retinoids (such as all-trans retinoic acids or a derivativesthereof); geldanamycin or a derivative thereof (such as 17-AAG), andother standard chemotherapeutic agents well recognized in the art.

In some variations, the chemotherapeutic agent is any of (and in somevariations selected from the group consisting of) adriamycin,colchicine, cyclophosphamide, actinomycin, bleomycin, duanorubicin,doxorubicin, epirubicin, mitomycin, methotrexate, mitoxantrone,fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin,etoposide, interferons, camptothecin and derivatives thereof,phenesterine, taxanes and derivatives thereof (e.g., paclitaxel andderivatives thereof, taxotere and derivatives thereof, and the like),topetecan, vinblastine, vincristine, tamoxifen, piposulfan, nab-5404,nab-5800, nab-5801, Irinotecan, HKP, Ortataxel, gemcitabine, Herceptin®,vinorelbine, Doxil®, capecitabine, Alimta®, Avastin®, Velcade®,Tarceva®, Neulasta®, Lapatinib, Sorafenib, derivatives thereof,chemotherapeutic agents known in the art, and the like. In somevariations, the chemotherapeutic agent is a composition comprisingnanoparticles comprising a thiocolchicine derivative and a carrierprotein (such as albumin).

In some variations, the chemotherapeutic agent is a antineoplastic agentincluding, but is not limited to, carboplatin, Navelbine® (vinorelbine),anthracycline (Doxil®), lapatinib (GW57016), Herceptin®, gemcitabine(Gemzar®), capecitabine (Xeloda®), Alimta®, cisplatin, 5-fluorouracil,epirubicin, cyclophosphamide, Avastin®, Velcade®, etc.

In some variations, the chemotherapeutic agent is an antagonist of otherfactors that are involved in tumor growth, such as EGFR, ErbB2 (alsoknown as Herb), ErbB3, ErbB4, or TNF. Sometimes, it may be beneficial toalso administer one or more cytokines to the individual. In somevariations, the therapeutic agent is a growth inhibitory agent. Suitabledosages for the growth inhibitory agent are those presently used and maybe lowered due to the combined action (synergy) of the growth inhibitoryagent and the taxane.

In some variations, the chemotherapeutic agent is a chemotherapeuticagent other than an anti-VEGF antibody, a HER2 antibody, interferon, andan HGFβ antagonist.

Reference to a chemotherapeutic agent herein applies to thechemotherapeutic agent or its derivatives and accordingly the inventioncontemplates and includes either of these variations (agent; agent orderivative(s)). “Derivatives” or “analogs” of a chemotherapeutic agentor other chemical moiety include, but are not limited to, compounds thatare structurally similar to the chemotherapeutic agent or moiety or arein the same general chemical class as the chemotherapeutic agent ormoiety. In some variations, the derivative or analog of thechemotherapeutic agent or moiety retains similar chemical and/orphysical property (including, for example, functionality) of thechemotherapeutic agent or moiety.

In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), and b) an effective amount of a tyrosine kinase inhibitor.In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising paclitaxel and an albumin (such asAbraxane®), and b) an effective amount of a tyrosine kinase inhibitor.Suitable tyrosine kinase inhibitors include, for example, imatinib(Gleevec®), gefitinib (Iressa®), Tarceva, Sutent® (sunitinib malate),and Lapatinib. In some variations, the tyrosine kinase inhibitor islapatinib. In some variations, the tyrosine kinase inhibitor is Tarceva.Tarceva is a small molecule human epidermal growth factor type1/epidermal growth factor receptor (HER1/EGFR) inhibitor whichdemonstrated, in a Phase III clinical trial, an increased survival inadvanced non-small cell lung cancer (NSCLC) individuals. In somevariations, the method is for treatment of breast cancer, includingtreatment of metastatic breast cancer and treatment of breast cancer ina neoadjuvant setting. In some variations, the method is for treatmentof advanced solid tumor. In some variations, there is provided a methodto inhibit the proliferation of EGFR expressing tumors in a mammalcomprising administering to a mammal infected with such tumors Abraxane®and gefitinib, wherein the gefitinib is administered by pulse-dosing.

In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), and b) an effective amount of an antimetabolite agent (suchas a nucleoside analog, including for example purine analogs andpyrimidine analogs). In some variations, the invention provides a methodof treating a proliferative disease (such as cancer) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising paclitaxel and analbumin (such as Abraxane®), and b) an effective amount of anantimetabolite agent. An “antimetabolic agent” is an agent which isstructurally similar to a metabolite, but cannot be used by the body ina productive manner. Many antimetabolite agents interfere withproduction of nucleic acids, RNA and DNA. For example, theantimetabolite can be a nucleoside analog, which includes, but is notlimited to, azacitidine, azathioprine, capecitabine (Xeloda®),cytarabine, cladribine, cytosine arabinoside (ara-C, cytosar),doxifluridine, fluorouracil (such as 5-fluorouracil), UFT, hydroxyurea,gemcitabine, mercaptopurine, methotrexate, thioguanine (such as6-thioguanine). Other anti-metabolites include, for example,L-asparaginase (Elspa), decarbazine (DTIC), 2-deoxy-D-glucose, andprocarbazine (matulane). In some variations, the nucleoside analog isany of (and in some variations selected from the group consisting of)gemcitabine, fluorouracil, and capecitabine. In some variations, themethod is for treatment of metastatic breast cancer or locally advancedbreast cancer. In some variations, the method is for first linetreatment of metastatic breast cancer. In some variations, the method isfor treatment of breast cancer in a neoadjuvant setting. In somevariations, the method is for treatment of any of NSCLC, metastaticcolorectal cancer, pancreatic cancer, or advanced solid tumor.

In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), and b) an effective amount of an alkylating agent. In somevariations, the invention provides a method of treating a proliferativedisease (such as cancer) in an individual, comprising administering tothe individual a) an effective amount of a composition comprisingnanoparticles comprising paclitaxel and an albumin (such as Abraxane®),and b) an effective amount of an alkylating agent. Suitable alkylatingagents include, but are not limited to, cyclophosphamide (Cytoxan),mechlorethamine, chlorambucil, melphalan, carmustine (BCNU), thiotepa,busulfan, alkyl sulphonates, ethylene imines, nitrogen mustard analogs,estramustine sodium phosphate, ifosfamide, nitrosoureas, lomustine, andstreptozocin. In some variations, the alkylating agent iscyclophosphamide. In some variations, the cyclophosphamide isadministered prior to the administration of the nanoparticlecomposition. In some variations, the method is for treatment of an earlystage breast cancer. In some variations, the method is for treatment ofa breast cancer in an adjuvant or a neoadjuvant setting.

In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), and b) an effective amount of a platinum-based agent. Insome variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising paclitaxel and an albumin (such asAbraxane®), and b) an effective amount of a platinum-based agent.Suitable platinum-based agents include, but are not limited to,carboplatin, cisplatin, and oxaliplatin. In some variations, theplatinum-based agent is carboplatin. In some variations, the method isfor treatment of: breast cancer (HER2 positive or HER2 negative,including metastatic breast cancer and advanced breast cancer); lungcancer (including advanced NSCLC, first line NSCLC, SCLC, and advancedsolid tumor malignancies in the lung); ovarian cancer; head and neckcancer; and melanoma (including metastatic melanoma).

In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), and b) an effective amount of an anthracycline antibiotic.In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising paclitaxel and an albumin (such asAbraxane®) and a carrier protein (such as albumin), and b) an effectiveamount of an anthracycline antibiotic. Suitable anthracycline antibioticinclude, but are not limited to, Doxil®, actinomycin, dactinomycin,daunorubicin (daunomycin), doxorubicin (adriamycin), epirubicin,idarubicin, mitoxantrone, valrubicin. In some variations, theanthracycline is any of (and in some variations selected from the groupconsisting of) Doxil®, epirubicin, and doxorubicin. In some variations,the method is for treatment of an early stage breast cancer. In somevariations, the method is for treatment of a breast cancer in anadjuvant or a neoadjuvant setting.

In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), and b) an effective amount of a vinca alkloid. In somevariations, the invention provides a method of treating a proliferativedisease (such as cancer) in an individual, comprising administering tothe individual a) an effective amount of a composition comprisingnanoparticles comprising palitaxel and an albumin (such as Abraxane®)and a carrier protein (such as albumin), and b) an effective amount of avinca alkloid. Suitable vinca alkaloids include, for example,vinblastine, vincristine, vindesine, vinorelbine (Navelbine®), andVP-16. In some variations, the vinca alkaloid is vinorelbine(Navelbine®). In some variations, the method is for treatment of stageIV breast cancer and lung cancer.

In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), and b) an effective amount of a macrolide. In somevariations, the invention provides a method of treating a proliferativedisease (such as cancer) in an individual, comprising administering tothe individual a) an effective amount of a composition comprisingnanoparticles comprising paclitaxel and an albumin (such as Abraxane®)and a carrier protein (such as albumin), and b) an effective amount of amacrolide. Suitable macrolides include, for example, rapamycin,carbomycin, and erythromycin. In some variations, the macrolide israpamycin or a derivative thereof. In some variations, the method is fortreatment of a solid tumor.

In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), and b) an effective amount of a topoisomerase inhibitor. Insome variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising paclitaxel and an albumin (such asAbraxane®) and a carrier protein (such as albumin), and b) an effectiveamount of a topoisomerase inhibitor. In some variations, thechemotherapeutic agent is a topoisomerase inhibitor, including, forexample, inhibitor of topoisomerase I and topoisomerase II. Exemplaryinhibitors of topoisomerase I include, but are not limited to,camptothecin, such as irinotecan and topotecan. Exemplary inhibitors oftopoisomerase II include, but are not limited to, amsacrine, etoposide,etoposide phosphate, and teniposide.

In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), and b) an effective amount of an antiangiogenic agent. Insome variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising paclitaxel and an albumin (such asAbraxane®) and a carrier protein (such as albumin), and b) an effectiveamount of an antiangiogenic agent. In some variations, the method is fortreatment of metastatic breast cancer, breast cancer in an adjuvantsetting or a neoadjuvant setting, lung cancer (such as first lineadvanced NSCLC and NSCLC), ovarian cancer, and melanoma (includingmetastatic melanoma).

Many anti-angiogenic agents have been identified and are known in theart, including those listed by Carmeliet and Jain (2000). Theanti-angiogenic agent can be naturally occurring or non-naturallyoccurring. In some variations, the chemotherapeutic agent is a syntheticantiangiogenic peptide. For example, it has been previously reportedthat the antiangiogenic activity of small synthetic pro-apoptic peptidescomprise two functional domains, one targeting the CD13 receptors(aminopeptidase N) on tumor microvessels and the other disrupting themitochondrial membrane following internalization. Nat. Med. 1999,5(9):1032-8. A second generation dimeric peptide, CNGRC-GG-d(KLAKLAK)₂,named HKP (Hunter Killer Peptide) was found to have improved antitumoractivity. Accordingly, in some variations, the antiangiogenic peptide isHKP. In some variations, the antiangiogenic agent is other than ananti-VEGF antibody (such as Avastin®).

In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), and b) an effective amount of a proteasome inhibitor, suchas bortezomib (Velcade). In some variations, the invention provides amethod of treating a proliferative disease (such as cancer) in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising paclitaxeland an albumin (such as Abraxane®) and a carrier protein (such asalbumin), and b) an effective amount of a proteasome inhibitor such asbortezomib (Velcade®).

In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), and b) an effective amount of a therapeutic antibody. Insome variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising paclitaxel and an albumin (such asAbraxane®) and a carrier protein (such as albumin), and b) an effectiveamount of a therapeutic antibody. Suitable therapeutic antibodiesinclude, but are not limited to, anti-VEGF antibody (such as Avastin®(bevacizumab)), anti-HER2 antibody (such as Herceptin® (trastuzumab)),Erbitux® (cetuximab), Campath (alemtuzumab), Myelotarg (gemtuzumab),Zevalin (ibritumomab tiuextan, Rituxan (rituximab), and Bexxar(tositumomab). In some variations, the chemotherapeutic agent isErbitux® (cetuximab). In some variations, the chemotherapeutic agent isa therapeutic antibody other than an antibody against VEGF or HER2. Insome variations, the method is for treatment of HER2 positive breastcancer, including treatment of advanced breast cancer, treatment ofmetastatic cancer, treatment of breast cancer in an adjuvant setting,and treatment of cancer in a neoadjuvant setting. In some variations,the method is for treatment of any of metastatic breast cancer, breastcancer in an adjuvant setting or a neoadjuvant setting, lung cancer(such as first line advanced NSCLC and NSCLC), ovarian cancer, head andneck cancer, and melanoma (including metastatic melanoma). For example,in some variations, there is provided a method for treatment of HER2positive metastatic breast cancer in an individual, comprisingadministering to the individual 125 mg/m² paclitaxel/albuminnanoparticle composition (such as Abraxane®) weekly for three weeks withthe fourth week off, concurrent with the administration of Herceptin®.

In some variations, there is provided a method of treating aproliferative disease (such as cancer, for example breast cancer) in anindividual, comprising administering to the individual: a) an effectiveamount of a composition comprising nanoparticles comprising taxane and acarrier protein, and b) an effective amount of an anti-VEGF antibody. Insome variations, the effective amounts of the taxane nanoparticlecomposition and the anti-VEGF antibody synergistically inhibit cellproliferation (such as tumor cell growth). In some variations, at leastabout 10% (including for example at least about any of 20%, 30%, 40%,60%, 70%, 80%, 90%, or 100%) cell proliferation is inhibited. In somevariations, the taxane is paclitaxel. In some variations, the anti-VEGFantibody is bevacizumab (such as Avastin®). In some variations, thetaxane is paclitaxel and the anti-VEGF antibody is bevacizumab (such asAvastin®). In some variations, the taxane in the nanoparticle in thecomposition is administered by intravenous administration. In somevariations, the anti-VEGF antibody is administered by intravenousadministration. In some variations, both the taxane in the nanoparticlecomposition and the anti-VEGF antibody are administered by intravenousadministration.

In some variations, there is provided a method of inhibiting tumormetastasis (such as metastasis of breast cancer) in an individual,comprising administering to the individual: a) an effective amount of acomposition comprising nanoparticles comprising taxane and a carrierprotein, and b) an effective amount of an anti-VEGF antibody. In somevariations, the effective amounts of the taxane nanoparticle compositionand the anti-VEGF antibody synergistically inhibit tumor metastasis. Insome variations, at least about 10% (including for example at leastabout any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) metastasis isinhibited. In some variations, method of inhibiting metastasis to lymphnode is provided. In some variations, method of inhibiting metastasis tothe lung is provided. In some variations, the taxane is paclitaxel. Insome variations, the anti-VEGF antibody is bevacizumab (such asAvastin®). In some variations, the taxane is paclitaxel and theanti-VEGF antibody is bevacizumab (such as Avastin®). In somevariations, the taxane in the nanoparticle in the composition isadministered by intravenous administration. In some variations, theanti-VEGF antibody is administered by intravenous administration. Insome variations, both the taxane in the nanoparticle composition and theanti-VEGF antibody are administered by intravenous administration.

Suitable dosages for anti-VEGF antibody include, for example, about 1mg/kg to about 20 mg/kg, including for example about 1 mg/kg to about 15mg/kg (such as about any of 2, 4, 6, 8, 10, or 12 mg/kg). In somevariations, the dosage of the anti-VEGF antibody is about 40 mg/m² toabout 600 mg/m², including for example about 100 mg/m² to about 400mg/m² (such as about any of 100, 200, or 300 mg/m²). In some variations,the taxane is paclitaxel. In some variations, the anti-VEGF antibody isbevacizumab (such as Avastin®). In some variations, the taxane ispaclitaxel and the anti-VEGF antibody is bevacizumab (such as Avastin®).

Suitable combinations of the amounts of taxane in a nanoparticlecomposition and the anti-VEGF antibody include, for example, about 1mg/kg to about 20 mg/kg (such as about any of 2, 5, 10, or 15 mg/kg)taxane in a nanoparticle composition and about 1 mg/kg to about 20 mg/kg(such as about any of 2, 4, 6, 8, 10, 12, 14, 16, or 18 mg/kg) anti-VEGFantibody; about 3 mg/m² to about 400 mg/m² (such as about any of 6, 10,15, 30, 45, 60, 100, 150, 200, or 300 mg/m²) taxane in a nanoparticlecomposition and 40 mg/m² to about 600 mg/m², including for example about100 mg/m² to about 400 mg/m² (such as about any of 100, 200, or 300mg/m²) anti-VEGF antibody; about 3 mg/m² to about 300 mg/m² (such asabout any of 6, 10, 15, 30, 45, 60, 100, 150, 200, or 300 mg/m²) taxanein a nanoparticle composition and about 1 mg/kg to about 20 mg/kg (suchas about any of 2, 4, 6, 8, 10, 12, 14, 16, or 18 mg/kg) anti-VEGFantibody. In some variations, the method comprises administering to anindividual at least about 200 mg/m² taxane in a nanoparticle compositionand at least about any of 2, 4, 8, or 10 mg/kg anti-VEGF antibody. Insome variations, the taxane is paclitaxel. In some variations, theanti-VEGF antibody is bevacizumab (such as Avastin®). In somevariations, the taxane is paclitaxel and the anti-VEGF antibody isbevacizumab (such as Avastin®).

In some variations of the method, the taxane nanoparticle compositionand the anti-VEGF antibody are administered simultaneously to theindividual. In some variations of the method, the administration of thenanoparticle composition and the chemotherapeutic agent are concurrent.One exemplary dosing regime for the combination therapy of taxanenanoparticle composition includes administration of 100 mg/m²-300 mg/m²(such as 200 mg/m²) taxane in nanoparticle composition at least weekly(including for example every 1, 2, 3, 4, 5, or 6 days) concurrent withadministration of 2 mg/kg -15 mg/kg (such as any of 4, 6, 8, 10 mg/kg or15 mg/kg) anti-VEGF antibody every two weeks or more frequently (forexample every week, twice every week, or three times a week). In somevariations, the taxane is paclitaxel. In some variations, the anti-VEGFantibody is bevacizumab (such as Avastin®). In some variations, thetaxane is paclitaxel and the anti-VEGF antibody is bevacizumab (such asAvastin®).

In some variations, the taxane nanoparticle composition and theanti-VEGF antibody are administered sequentially to the individual. Forexample, in some variations, the taxane nanoparticle composition isadministered for at least one (such as at least any of two, three, four,five, or six) cycles prior to the administration of the anti-VEGFantibody. This is then followed by the administration of an anti-VEGFantibody for at least once (such as twice) a week for at least about 3(such as 4, 5, or 6) weeks. One exemplary dosing regime for thecombination therapy of taxane nanoparticle composition (such aspaclitaxel/albumin nanoparticle composition, for example Abraxane®) andanti-VEGF antibody (such as bevacizumab, for example Avastin®) includesadministration of 10 mg/kg taxane in a nanoparticle composition dailyfor 5 days in two cycles separated by one week followed byadministration of an anti-VEGF antibody at dosages of 2 mg/kg, 4 mg/kg,or 8 mg/kg twice a week for 6 weeks. In some variations, the taxane ispaclitaxel. In some variations, the anti-VEGF antibody is bevacizumab(such as Avastin®). In some variations, the taxane is paclitaxel and theanti-VEGF antibody is bevacizumab (such as Avastin®).

In some variations, the effective amount of Abraxane is between about 80mg/m² to about 150 mg/m² and the effective amount of bevacizumab isabout 10 mg/kg or about 15 mg/kg. In some variations, the effectiveamount of Abraxane is about 100 mg/m². In some variations, Abraxane isadministered weekly. In some variations, bevacizumab is administeredevery 2 weeks or every 3 weeks.

In some variations, the effective amount of Abraxane is between about200 mg/m² to about 350 mg/m² and the effective amount of bevacizumab isbetween about 5 mg/kg and about 15 mg/kg. In some variations, theeffective amount of Abraxane is about 260 mg/m². In some variations,Abraxane is administered every three weeks. In some variations, theeffective amount of bevacizumab is between about 5 mg/kg and about 10mg/kg. In some variations, the effective amount of bevacizumab is about15 mg/kg. In some variations, bevacizumab is administered every 2 weeksor every 3 weeks.

In some variations, there is provided a method of treating aproliferative disease (such as cancer, for example breast cancer) in anindividual comprising administering to the individual: (1) an effectiveamount of a composition comprising nanoparticles comprising a taxane anda carrier protein, and (2) an effective amount of an anti-VEGF antibody,wherein the effective amount of taxane in the nanoparticle compositionis between about 45 mg/m² to about 350 mg/m² and the effective amount ofanti-VEGF antibody is greater than 1 mg/kg to less than 10 mg/kg orgreater than 15 mg/kg to less than 20 mg/kg. In some variations, theeffective amount of taxane in the nanoparticle composition is betweenabout 80 mg/m² to about 150 mg/m² and the effective amount of anti-VEGFantibody is greater than 1 mg/kg to less than 10 mg/kg or greater than15 mg/kg to less than 20 mg/kg. In some variations, the effective amountof taxane in the nanoparticle composition is about 100 mg/m². In somevariations, the taxane in a nanoparticle composition is administeredweekly. In some variations, the anti-VEGF antibody is administered every2 weeks or every 3 weeks. In some variations, the effective amount oftaxane in the nanoparticle composition is between about 170 mg/m² toabout 200 mg/m² and the effective amount of anti-VEGF antibody isgreater than 1 mg/kg to less than 10 mg/kg or greater than 15 mg/kg toless than 20 mg/kg. In some variations, the taxane in a nanoparticlecomposition is administered every two weeks. In some variations, theeffective amount of taxane in the nanoparticle composition is betweenabout 200 mg/m² to about 350 mg/m² and the effective amount of anti-VEGFantibody is greater than 1 mg/kg to less than 10 mg/kg or greater than15 mg/kg to less than 20 mg/kg. In some variations, the effective amountof taxane in the nanoparticle composition is about 260 mg/m². In somevariations, the taxane in a nanoparticle composition is administeredevery three weeks. In some variations, the anti-VEGF antibody isadministered every 2 weeks or every 3 weeks. In some variations of anyof the above methods, the effective amount of anti-VEGF antibody isabout 2 mg/kg, about 4 mg/kg, about 6 mg/kg, or about 8 mg/kg.

In some variations, the taxane is paclitaxel. In some variations, thecarrier protein is albumin. In some variations, the taxane is paclitaxeland the carrier protein is albumin. In some variations, the albumin ishuman serum albumin. In some variations, the nanoparticles comprising ataxane and a carrier protein is Abraxane. In some variations, theanti-VEGF antibody is bevacizumab (i.e., Avastin®). In some variations,the nanoparticles comprising a taxane and a carrier protein is Abraxaneand the anti-VEGF antibody is bevacizumab (i.e., Avastin®). In somevariations, the effective amount of Abraxane is between about 45 mg/m²to about 350 mg/m² and the effective amount of bevacizumab is greaterthan 1 mg/kg to less than 10 mg/kg or greater than 15 mg/kg to less than20 mg/kg. In some variations, the effective amount of Abraxane isbetween about 80 mg/m² to about 150 mg/m² and the effective amount ofbevacizumab is greater than 1 mg/kg to less than 10 mg/kg or greaterthan 15 mg/kg to less than 20 mg/kg. In some variations, the effectiveamount of Abraxane is about 100 mg/m². In some variations, Abraxane isadministered weekly. In some variations, the bevacizumab is administeredevery 2 weeks or every 3 weeks. In some variations, the effective amountof Abraxane is between about 170 mg/m² to about 200 mg/m² and theeffective amount of bevacizumab is greater than 1 mg/kg to less than 10mg/kg or greater than 15 mg/kg to less than 20 mg/kg. In somevariations, the Abraxane is administered every two weeks. In somevariations, the effective amount of Abraxane is between about 200 mg/m²to about 350 mg/m² and the effective amount of bevacizumab is greaterthan 1 mg/kg to less than 10 mg/kg or greater than 15 mg/kg to less than20 mg/kg. In some variations, the effective amount of Abraxane is about260 mg/m². In some variations, Abraxane is administered every threeweeks. In some variations, the bevacizumab is administered every 2 weeksor every 3 weeks. In some variations of any of the above methods, theeffective amount of bevacizumab is about 2 mg/kg, about 4 mg/kg, about 6mg/kg, or about 8 mg/kg.

In some variations of any of the above methods, the proliferativedisease is cancer. In some variations, the cancer is breast cancer, lungcancer, prostate cancer, ovarian cancer, or melanoma. In somevariations, the breast cancer is metastatic breast cancer. In somevariations, the metastatic breast cancer is heavily pretreated withanthracylines and/or taxanes. In some variations, the lung cancer isnon-small cell lung cancer (NSCLC). In some variations, the NSCLC isstage IIIB and/or stage IV non-small cell lung cancer.

In some variations, there is provided a method of inhibiting tumormetastasis (such as metastasis of breast cancer) in an individualcomprising administering to the individual: (1) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein, and (2) an effective amount of an anti-VEGF antibody, whereinthe effective amount of taxane in the nanoparticle composition isbetween about 45 mg/m² to about 350 mg/m² and the effective amount ofanti-VEGF antibody is greater than 1 mg/kg to less than 10 mg/kg orgreater than 15 mg/kg to less than 20 mg/kg. In some variations, theeffective amount of taxane in the nanoparticle composition is betweenabout 80 mg/m² to about 150 mg/m² and the effective amount of anti-VEGFantibody is greater than 1 mg/kg to less than 10 mg/kg or greater than15 mg/kg to less than 20 mg/kg. In some variations, the effective amountof taxane in the nanoparticle composition is about 100 mg/m². In somevariations, the taxane in a nanoparticle composition is administeredweekly. In some variations, the anti-VEGF antibody is administered every2 weeks or every 3 weeks. In some variations, the effective amount oftaxane in the nanoparticle composition is between about 170 mg/m² toabout 200 mg/m² and the effective amount of anti-VEGF antibody isgreater than 1 mg/kg to less than 10 mg/kg or greater than 15 mg/kg toless than 20 mg/kg. In some variations, the taxane in a nanoparticlecomposition is administered every two weeks. In some variations, theeffective amount of taxane in the nanoparticle composition is betweenabout 200 mg/m² to about 350 mg/m² and the effective amount of anti-VEGFantibody is greater than 1 mg/kg to less than 10 mg/kg or greater than15 mg/kg to less than 20 mg/kg. In some variations, the effective amountof taxane in the nanoparticle composition is about 260 mg/m². In somevariations, the taxane in a nanoparticle composition is administeredevery three weeks. In some variations, the anti-VEGF antibody isadministered every 2 weeks or every 3 weeks. In some variations of anyof the above methods, the effective amount of anti-VEGF antibody isabout 2 mg/kg, about 4 mg/kg, about 6 mg/kg, or about 8 mg/kg.

In some variations, the taxane is paclitaxel. In some variations, thecarrier protein is albumin. In some variations, the taxane is paclitaxeland the carrier protein is albumin. In some variations, the albumin ishuman serum albumin. In some variations, the nanoparticles comprising ataxane and a carrier protein is Abraxane. In some variations, theanti-VEGF antibody is bevacizumab (i.e., Avastin®). In some variations,the nanoparticles comprising a taxane and a carrier protein is Abraxaneand the anti-VEGF antibody is bevacizumab (i.e., Avastin®). In somevariations, the effective amount of Abraxane is between about 45 mg/m²to about 350 mg/m² and the effective amount of bevacizumab is greaterthan 1 mg/kg to less than 10 mg/kg or greater than 15 mg/kg to less than20 mg/kg. In some variations, the effective amount of Abraxane isbetween about 80 mg/m² to about 150 mg/m² and the effective amount ofbevacizumab is greater than 1 mg/kg to less than 10 mg/kg or greaterthan 15 mg/kg to less than 20 mg/kg. In some variations, the effectiveamount of Abraxane is about 100 mg/m². In some variations, Abraxane isadministered weekly. In some variations, the bevacizumab is administeredevery 2 weeks or every 3 weeks. In some variations, the effective amountof Abraxane is between about 170 mg/m² to about 200 mg/m² and theeffective amount of bevacizumab is greater than 1 mg/kg to less than 10mg/kg or greater than 15 mg/kg to less than 20 mg/kg. In somevariations, the Abraxane is administered every two weeks. In somevariations, the effective amount of Abraxane is between about 200 mg/m²to about 350 mg/m² and the effective amount of bevacizumab is greaterthan 1 mg/kg to less than 10 mg/kg or greater than 15 mg/kg to less than20 mg/kg. In some variations, the effective amount of Abraxane is about260 mg/m². In some variations, Abraxane is administered every threeweeks. In some variations, the bevacizumab is administered every 2 weeksor every 3 weeks. In some variations of any of the above methods, theeffective amount of bevacizumab is about 2 mg/kg, about 4 mg/kg, about 6mg/kg, or about 8 mg/kg.

In some variations of any of the above methods, the tumor metastasis ismetastatic breast cancer, metastatic lung cancer, metastatic prostatecancer, metastatic ovarian cancer, or metastatic melanoma. In somevariations, tumor metastasis is metastatic breast cancer. In somevariations, the metastatic breast cancer is heavily pretreated withanthracylines and/or taxanes. In some variations, the metastatic lungcancer is metastatic non-small cell lung cancer (NSCLC).

In some variations, there is provided a method of treating aproliferative disease (such as cancer, for example breast cancer) in anindividual comprising administering to the individual: (1) an effectiveamount of a composition comprising a taxane, and (2) an effective amountof an anti-VEGF antibody, wherein the effective amount of the anti-VEGFantibody is an amount effective to suppress taxane-mediated induction ofVEGF in vivo. In some variations, the taxane-mediated induction of VEGFin vivo is taxane-mediated induction of VEGF-A. In some variations, thetaxane is paclitaxel. In some variations, the taxane is docetaxel. Insome variations, the composition comprising a taxane is a compositioncomprising nanoparticles comprising a taxane and a carrier protein. Insome variations, the nanoparticles comprising a taxane are nanoparticlescomprising paclitaxel. In some variations, the nanoparticles comprisinga taxane are nanoparticles comprising docetaxel. In some variations, thecarrier protein is albumin. In some variations, the taxane is paclitaxeland the carrier protein is albumin. In some variations, the albumin ishuman serum albumin. In some variations, the nanoparticles comprising ataxane and a carrier protein is Abraxane. In some variations, theanti-VEGF antibody is bevacizumab (i.e., Avastin®). In some variations,the nanoparticles comprising a taxane and a carrier protein is Abraxaneand the anti-VEGF antibody is bevacizumab (i.e., Avastin®). In somevariations, the effective amounts of the taxane and the anti-VEGFantibody synergistically inhibit cell proliferation (such as tumor cellgrowth). In some variations, at least about 10% (including for exampleat least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) cellproliferation is inhibited. In some variations, the taxane isadministered by intravenous administration. In some variations, theanti-VEGF antibody is administered by intravenous administration. Insome variations, both the taxane and the anti-VEGF antibody areadministered by intravenous administration.

In some variations, there is provided a method of inhibiting tumormetastasis (such as metastasis of breast cancer) in an individual,comprising administering to the individual: (1) an effective amount of acomposition comprising a taxane, and (2) an effective amount of ananti-VEGF antibody, wherein the effective amount of the anti-VEGFantibody is an amount effective to suppress taxane-mediated induction ofVEGF in vivo. In some variations, the taxane-mediated induction of VEGFin vivo is taxane-mediated induction of VEGF-A. In some variations, thetaxane is paclitaxel. In some variations, the taxane is docetaxel. Insome variations, the composition comprising a taxane is a compositioncomprising nanoparticles comprising a taxane and a carrier protein. Insome variations, the nanoparticles comprising a taxane are nanoparticlescomprising paclitaxel. In some variations, the nanoparticles comprisinga taxane are nanoparticles comprising docetaxel. In some variations, thecarrier protein is albumin. In some variations, the taxane is paclitaxeland the carrier protein is albumin. In some variations, the albumin ishuman serum albumin. In some variations, the nanoparticles comprising ataxane and a carrier protein is Abraxane. In some variations, theanti-VEGF antibody is bevacizumab (i.e., Avastin®). In some variations,the nanoparticles comprising a taxane and a carrier protein is Abraxaneand the anti-VEGF antibody is bevacizumab (i.e., Avastin®). In somevariations, at least about 10% (including for example at least about anyof 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) metastasis is inhibited.In some variations, method of inhibiting metastasis to lymph node isprovided. In some variations, method of inhibiting metastasis to thelung is provided. In some variations, the taxane is administered byintravenous administration. In some variations, the anti-VEGF antibodyis administered by intravenous administration. In some variations, boththe taxane and the anti-VEGF antibody are administered by intravenousadministration.

Suitable dosages for anti-VEGF antibody include, for example, about 1mg/kg to about 20 mg/kg, including for example about 1 mg/kg to about 15mg/kg (such as about any of 2, 4, 6, 8, 10, or 12 mg/kg). In somevariations, the dosage of the anti-VEGF antibody is about 40 mg/m² toabout 600 mg/m², including for example about 100 mg/m² to about 400mg/m² (such as about any of 100, 200, or 300 mg/m²). In some variations,the taxane is paclitaxel. In some variations, the taxane is docetaxel.In some variations, the anti-VEGF antibody is bevacizumab (such asAvastin®). In some variations, the anti-VEGF antibody is bevacizumab(such as Avastin®) and the taxane is paclitaxel.

Suitable combinations of the amounts of taxane and the anti-VEGFantibody include, for example, about 1 mg/kg to about 20 mg/kg (such asabout any of 2, 5, 10, or mg/kg) taxane and about 1 mg/kg to about 20mg/kg (such as about any of 2, 4, 6, 8, 10, 12, 14, 16, or 18 mg/kg)anti-VEGF antibody; about 3 mg/m² to about 400 mg/m² (such as about anyof 6, 10, 15, 30, 45, 60, 100, 150, 200, or 300 mg/m²) taxane and 40mg/m² to about 600 mg/m², including for example about 100 mg/m² to about400 mg/m² (such as about any of 100, 200, or 300 mg/m²) anti-VEGFantibody; about 3 mg/m² to about 300 mg/m² (such as about any of 6, 10,15, 30, 45, 60, 100, 150, 200, or 300 mg/m²) taxane and about 1 mg/kg toabout 20 mg/kg (such as about any of 2, 4, 6, 8, 10, 12, 14, 16, or 18mg/kg) anti-VEGF antibody. In some variations, the method comprisesadministering to an individual at least about 200 mg/m² taxane and atleast about any of 2, 4, 8, or 10 mg/kg anti-VEGF antibody. In somevariations, the taxane is paclitaxel. In some variations, the taxane isdocetaxel. In some variations, the anti-VEGF antibody is bevacizumab(such as Avastin®). In some variations, the anti-VEGF antibody isbevacizumab (such as Avastin®) and the taxane is paclitaxel.

In some variations of the methods, the taxane and the anti-VEGF antibodyare administered simultaneously to the individual. In some variations ofthe methods, the administration of the taxane and the anti-VEGF antibodyare concurrent. One exemplary dosing regime for the combination therapyof taxane (such as paclitaxel) includes administration of 100 mg/m²-300mg/m² (such as 200 mg/m²) taxane at least weekly (including for exampleevery 1, 2, 3, 4, 5, or 6 days) concurrent with administration of 2mg/kg -15 mg/kg (such as any of 4, 6, 8, 10 mg/kg or 15 mg/kg) anti-VEGFantibody every two weeks or more frequently (for example every week,twice every week, or three times a week). In some variations, the taxaneis paclitaxel. In some variations, the taxane is docetaxel. In somevariations, the anti-VEGF antibody is bevacizumab (such as Avastin®). Insome variations, the anti-VEGF antibody is bevacizumab (such asAvastin®) and the taxane is paclitaxel.

In some variations, the taxane and the anti-VEGF antibody areadministered sequentially to the individual. For example, in somevariations, the taxane is administered for at least one (such as atleast any of two, three, four, five, or six) cycles prior to theadministration of the anti-VEGF antibody. This is then followed by theadministration of an anti-VEGF antibody for at least once (such astwice) a week for at least about 3 (such as 4, 5, or 6) weeks. Oneexemplary dosing regime for the combination therapy of taxanecomposition (such as paclitaxel/albumin nanoparticle composition, forexample Abraxane®) and anti-VEGF antibody (such as bevacizumab, forexample Avastin®) includes administration of 10 mg/kg taxane in ananoparticle composition daily for 5 days in two cycles separated by oneweek followed by administration of an anti-VEGF antibody at dosages of 2mg/kg, 4 mg/kg, or 8 mg/kg twice a week for 6 weeks. In some variations,the taxane is paclitaxel. In some variations, the taxane is docetaxel.In some variations, the anti-VEGF antibody is bevacizumab (such asAvastin®). In some variations, the anti-VEGF antibody is bevacizumab(such as Avastin®) and the taxane is paclitaxel.

In some variations, the effective amount of taxane in the composition isbetween about 45 mg/m² to about 350 mg/m² and the effective amount ofanti-VEGF antibody is between about 1 mg/kg to about 20 mg/kg. In somevariations, the effective amount of taxane in the composition is betweenabout 80 mg/m² to about 150 mg/m² and the effective amount of anti-VEGFantibody is between about 1 mg/kg to about 20 mg/kg. In some variations,the effective amount of taxane in the composition is about 100 mg/m². Insome variations, the taxane is administered weekly. In some variations,the effective amount of taxane in the composition is between about 170mg/m² to about 200 mg/m² and the effective amount of anti-VEGF antibodyis between about 1 mg/kg to about 20 mg/kg. In some variations, theeffective amount of taxane in the composition is between about 200 mg/m²to about 350 mg/m² and the effective amount of anti-VEGF antibody isbetween about 1 mg/kg to about 20 mg/kg. In some variations, the taxaneis administered every two weeks. In some variations, the effectiveamount of taxane in the composition is about 260 mg/m². In somevariations, the taxane is administered every three weeks. In somevariations of any of the above methods, the effective amount ofanti-VEGF antibody is greater than 1 mg/kg to less than 10 mg/kg orgreater than 15 mg/kg to less than 20 mg/kg. In some variations, theeffective amount of anti-VEGF antibody is between about 5 to about 10mg/kg. In some variations of any of the above methods, the effectiveamount of anti-VEGF antibody is about 2 mg/kg, about 4 mg/kg, about 6mg/kg, about 8 mg/kg, about 10 mg/kg, about 12 mg/kg, or about 15 mg/kg.In some variations, the effective amount of anti-VEGF antibody is about10 mg/kg. In some variations, the effective amount of anti-VEGF antibodyis about 15 mg/kg. In some variations, the anti-VEGF antibody isadministered every two weeks or every three weeks. In some variations,the taxane is paclitaxel. In some variations, the taxane is docetaxel.In some variations, the anti-VEGF antibody is bevacizumab (such asAvastin®). In some variations, the anti-VEGF antibody is bevacizumab(such as Avastin®) and the taxane is paclitaxel.

In some variations, there is provided a method of treating aproliferative disease (such as cancer, for example breast cancer) in anindividual comprising administering to the individual: (1) an effectiveamount of a composition comprising nanoparticles comprising a taxane anda carrier protein, and (2) an effective amount of an anti-VEGF antibody,wherein the effective amount of the anti-VEGF antibody is an amounteffective to suppress taxane-mediated induction of VEGF in vivo. In somevariations, the taxane-mediated induction of VEGF in vivo istaxane-mediated induction of VEGF-A. In some variations, the effectiveamount of taxane in the nanoparticle composition is between about 45mg/m² to about 350 mg/m² and the effective amount of anti-VEGF antibodyis between about 1 mg/kg to about 20 mg/kg. In some variations, theeffective amount of taxane in the nanoparticle composition is betweenabout 80 mg/m² to about 150 mg/m² and the effective amount of anti-VEGFantibody is between about 1 mg/kg to about 20 mg/kg. In some variations,the effective of amount of taxane in the nanoparticle composition isabout 100 mg/m². In some variations, the taxane in a nanoparticlecomposition is administered weekly. In some variations, the effectiveamount of taxane in the nanoparticle composition is between about 170mg/m² to about 200 mg/m² and the effective amount of anti-VEGF antibodyis between about 1 mg/kg to about 20 mg/kg. In some variations, thetaxane in a nanoparticle composition is administered every two weeks. Insome variations, the effective amount of taxane in the nanoparticlecomposition is between about 200 mg/m² to about 350 mg/m² and theeffective amount of anti-VEGF antibody is between about 1 mg/kg to about20 mg/kg. In some variations, the effective amount of taxane in thenanoparticle composition is about 260 mg/m². In some variations, taxanein the nanoparticle composition is administered every three weeks. Insome variations, the anti-VEGF antibody is administered every 2 weeks orevery 3 weeks. In some variations of any of the above methods, theeffective amount of anti-VEGF antibody is greater than 1 mg/kg to lessthan 10 mg/kg or greater than 15 mg/kg to less than 20 mg/kg. In somevariations, the effective amount of anti-VEGF antibody is between about5 mg/kg and about 10 mg/kg. In some variations, the effective amount ofanti-VEGF antibody is about 2 mg/kg, about 4 mg/kg, about 6 mg/kg, about8 mg/kg, about 10 mg/kg, about 12 mg/kg, or about 15 mg/kg. In somevariations, the effective amount of anti-VEGF antibody is about 10mg/kg. In some variations, the effective amount of anti-VEGF antibody isabout 15 mg/kg.

In some variations, the taxane is paclitaxel. In some variations, thecarrier protein is albumin. In some variations, the taxane is paclitaxeland the carrier protein is albumin. In some variations, the albumin ishuman serum albumin. In some variations, the nanoparticles comprising ataxane and a carrier protein is Abraxane. In some variations, theanti-VEGF antibody is bevacizumab (i.e., Avastin®). In some variations,the nanoparticles comprising a taxane and a carrier protein is Abraxaneand the anti-VEGF antibody is bevacizumab (i.e., Avastin®). In somevariations, the effective amount of Abraxane is between about 45 mg/m²to about 350 mg/m² and the effective amount of bevacizumab is betweenabout 1 mg/kg to about 20 mg/kg. In some variations, the effectiveamount of Abraxane is between about 80 mg/m² to about 150 mg/m² and theeffective amount of bevacizumab is between about 1 mg/kg to about 20mg/kg. In some variations, the effective of amount of Abraxane is about100 mg/m². In some variations, Abraxane is administered weekly. In somevariations, the effective amount of Abraxane is between about 170 mg/m²to about 200 mg/m² and the effective amount of bevacizumab is betweenabout 1 mg/kg to about 20 mg/kg. In some variations, Abraxane isadministered every two weeks. In some variations, the effective amountof Abraxane is between about 200 mg/m² to about 350 mg/m² and theeffective amount of bevacizumab is between about 1 mg/kg to about 20mg/kg. In some variations, the effective amount of Abraxane is about 260mg/m². In some variations, Abraxane is administered every three weeks.In some variations, bevacizumab is administered every 2 weeks or every 3weeks. In some variations of any of the above methods, the effectiveamount of bevacizumab is greater than 1 mg/kg to less than 10 mg/kg orgreater than 15 mg/kg to less than 20 mg/kg. In some variations, theeffective amount of bevacizumab is between about 5 mg/kg and about 10mg/kg. In some variations, the effective amount of bevacizumab is about2 mg/kg, about 4 mg/kg, about 6 mg/kg, about 8 mg/kg, about 10 mg/kg,about 12 mg/kg, or about 15 mg/kg. In some variations, the effectiveamount of bevacizumab is about 10 mg/kg. In some variations, theeffective amount of bevacizumab is about 15 mg/kg.

In some variations, the effective amount of Abraxane is between about 80mg/m² to about 150 mg/m² and the effective amount of bevacizumab isabout 10 mg/kg or about 15 mg/kg. In some variations, the effectiveamount of Abraxane is about 100 mg/m². In some variations, Abraxane isadministered weekly. In some variations, bevacizumab is administeredevery 2 weeks or every 3 weeks.

In some variations, the effective amount of Abraxane is between about200 mg/m² to about 350 mg/m² and the effective amount of bevacizumab isbetween about 5 mg/kg and about 15 mg/kg. In some variations, theeffective amount of Abraxane is about 260 mg/m². In some variations,Abraxane is administered weekly. In some variations, the effectiveamount of bevacizumab is between about 5 mg/kg and about 10 mg/kg. Insome variations, the effective amount of bevacizumab is about 15 mg/kg.In some variations, bevacizumab is administered every 2 weeks or every 3weeks.

In some variations of any of the above methods, the proliferativedisease is cancer. In some variations, the cancer is breast cancer, lungcancer, prostate cancer, ovarian cancer, or melanoma. In somevariations, the breast cancer is metastatic breast cancer. In somevariations, the metastatic breast cancer is heavily pretreated withanthracylines and/or taxanes. In some variations, the lung cancer isnon-small cell lung cancer (NSCLC). In some variations, the NSCLC isstage IIIB and/or stage IV non-small cell lung cancer.

In some variations, there is provided a method of inhibiting tumormetastasis (such as metastasis of breast cancer) in an individualcomprising administering to the individual: (1) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein, and (2) an effective amount of an anti-VEGF antibody, whereinthe effective amount of the anti-VEGF antibody is an amount effective tosuppress taxane-mediated induction of VEGF in vivo. In some variations,the taxane-mediated induction of VEGF in vivo is taxane-mediatedinduction of VEGF-A. In some variations, the effective amount of taxanein the nanoparticle composition is between about 45 mg/m² to about 350mg/m² and the effective amount of anti-VEGF antibody is between about 1mg/kg to about 20 mg/kg. In some variations, the effective amount oftaxane in the nanoparticle composition is between about 80 mg/m² toabout 150 mg/m² and the effective amount of anti-VEGF antibody isbetween about 1 mg/kg to about 20 mg/kg. In some variations, theeffective of amount of taxane in the nanoparticle composition is about100 mg/m². In some variations, the taxane in a nanoparticle compositionis administered weekly. In some variations, the effective amount oftaxane in the nanoparticle composition is between about 170 mg/m² toabout 200 mg/m² and the effective amount of anti-VEGF antibody isbetween about 1 mg/kg to about 20 mg/kg. In some variations, the taxanein a nanoparticle composition is administered every two weeks. In somevariations, the effective amount of taxane in the nanoparticlecomposition is between about 200 mg/m² to about 350 mg/m² and theeffective amount of anti-VEGF antibody is between about 1 mg/kg to about20 mg/kg. In some variations, the effective amount of taxane in thenanoparticle composition is about 260 mg/m². In some variations, taxanein the nanoparticle composition is administered every three weeks. Insome variations, the anti-VEGF antibody is administered every 2 weeks orevery 3 weeks. In some variations of any of the above methods, theeffective amount of anti-VEGF antibody is greater than 1 mg/kg to lessthan 10 mg/kg or greater than 15 mg/kg to less than 20 mg/kg. In somevariations, the effective amount of anti-VEGF antibody is between about5 mg/kg and about 10 mg/kg. In some variations, the effective amount ofanti-VEGF antibody is about 2 mg/kg, about 4 mg/kg, about 6 mg/kg, about8 mg/kg, about 10 mg/kg, about 12 mg/kg, or about 15 mg/kg. In somevariations, the effective amount of anti-VEGF antibody is about 10mg/kg. In some variations, the effective amount of anti-VEGF antibody isabout 15 mg/kg.

In some variations, the taxane is paclitaxel. In some variations, thecarrier protein is albumin. In some variations, the taxane is paclitaxeland the carrier protein is albumin. In some variations, the albumin ishuman serum albumin. In some variations, the nanoparticles comprising ataxane and a carrier protein is Abraxane. In some variations, theanti-VEGF antibody is bevacizumab (i.e., Avastin®). In some variations,the nanoparticles comprising a taxane and a carrier protein is Abraxaneand the anti-VEGF antibody is bevacizumab (i.e., Avastin®). In somevariations, the effective amount of Abraxane is between about 45 mg/m²to about 350 mg/m² and the effective amount of bevacizumab is betweenabout 1 mg/kg to about 20 mg/kg. In some variations, the effectiveamount of Abraxane is between about 80 mg/m² to about 150 mg/m² and theeffective amount of bevacizumab is between about 1 mg/kg to about 20mg/kg. In some variations, the effective of amount of Abraxane is about100 mg/m². In some variations, Abraxane is administered weekly. In somevariations, the effective amount of Abraxane is between about 170 mg/m²to about 200 mg/m² and the effective amount of bevacizumab is betweenabout 1 mg/kg to about 20 mg/kg. In some variations, Abraxane isadministered every two weeks. In some variations, the effective amountof Abraxane is between about 200 mg/m² to about 350 mg/m² and theeffective amount of bevacizumab is between about 1 mg/kg to about 20mg/kg. In some variations, the effective amount of Abraxane is about 260mg/m². In some variations, Abraxane is administered every three weeks.In some variations, bevacizumab is administered every 2 weeks or every 3weeks. In some variations of any of the above methods, the effectiveamount of bevacizumab is greater than 1 mg/kg to less than 10 mg/kg orgreater than 15 mg/kg to less than 20 mg/kg. In some variations, theeffective amount of bevacizumab is between about 5 mg/kg and about 10mg/kg. In some variations, the effective amount of bevacizumab is about2 mg/kg, about 4 mg/kg, about 6 mg/kg, about 8 mg/kg, about 10 mg/kg,about 12 mg/kg, or about 15 mg/kg. In some variations, the effectiveamount of bevacizumab is about 10 mg/kg. In some variations, theeffective amount of bevacizumab is about 15 mg/kg.

In some variations, the effective amount of Abraxane is between about 80mg/m² to about 150 mg/m² and the effective amount of bevacizumab isabout 10 mg/kg or about 15 mg/kg. In some variations, the effectiveamount of Abraxane is about 100 mg/m². In some variations, Abraxane isadministered weekly. In some variations, bevacizumab is administeredevery 2 weeks or every 3 weeks.

In some variations, the effective amount of Abraxane is between about200 mg/m² to about 350 mg/m² and the effective amount of bevacizumab isbetween about 5 mg/kg and about 15 mg/kg. In some variations, theeffective amount of Abraxane is about 260 mg/m². In some variations,Abraxane is administered weekly. In some variations, the effectiveamount of bevacizumab is between about 5 mg/kg and about 10 mg/kg. Insome variations, the effective amount of bevacizumab is about 15 mg/kg.In some variations, bevacizumab is administered every 2 weeks or every 3weeks.

In some variations of any of the above methods, the tumor metastasis ismetastatic breast cancer, metastatic lung cancer, metastatic prostatecancer, metastatic ovarian cancer, or metastatic melanoma. In somevariations, tumor metastasis is metastatic breast cancer. In somevariations, the metastatic breast cancer is heavily pretreated withanthracylines and/or taxanes. In some variations, the metastatic lungcancer is metastatic non-small cell lung cancer (NSCLC).

The term “amount effective to suppress taxane-mediated induction of VEGFin vivo,” as used herein, refers to and includes both complete(including substantially complete) and/or partial suppression. Methodsindicating such suppression are known in the art and described herein,although it is understood that when administering to an individualpatient based on established medical practice on the basis of clinicaltrials, such measurements need not be given in an individual. In somevariations, the term “amount effective to suppress taxane-mediatedinduction of VEGF,” as used herein, refers to substantially completeprevention of VEGF expression and/or activity or reduction in the amountVEGF (such as VEGF-A) expression and/or activity in cells, tissues orfluids in vivo upon administration of a formulation containing a taxane.In some variations, the reduction in the amount VEGF expression and/oractivity in cells, tissues or fluids in vivo upon administration of aformulation containing a taxane is by at least about any of 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. In some variations, thesuppression of taxane induction can be observed qualitatively and/orquantitatively by methods known in the art and described herein.

In some variations, two or more chemotherapeutic agents are administeredin addition to the taxane in the nanoparticle composition. These two ormore chemotherapeutic agents may (but not necessarily) belong todifferent classes of chemotherapeutic agents. Examples of thesecombinations are provided herein. Other combinations are alsocontemplated.

In some variations, there is provided a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), b) an effective amount of an antimetabolite (such as anucleoside analog, for example, gemcitabine), and c) an anthracyclineantibiotic (such as epirubicin). In some variations, there is provided amethod of treating a proliferative disease (such as cancer) in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising paclitaxeland an albumin (such as Abraxane®), b) an effective amount of anantimetabolite (such as a nucleoside analog, for example, gemcitabine),and c) an effective amount of an anthracycline antibiotic (such asepirubicin). In some variations, the method is for treatment of breastcancer in a neoadjuvant setting. For example, in some variations, thereis provided a method of treating locally advanced/inflammatory cancer inan individual comprising administering to the individual 220 mg/m²paclitaxel/albumin nanoparticle composition (such as Abraxane®) everytwo weeks; 2000 mg/m² gemcitabine, every two weeks; and 50 mg/m²epirubicin, every two weeks. In some variations, there is provided amethod of treating breast cancer in an individual in an adjuvantsetting, comprising administering to the individual 175 mg/m²paclitaxel/albumin nanoparticle composition (such as Abraxane®) everytwo weeks, 2000 mg/m² gemcitabine, every two weeks, and 50 mg/m²epirubicin, every two weeks.

In some variations, there is provided a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), b) an effective amount of a platinum-based agent (such ascarboplatin), and c) a therapeutic antibody (such as ant-HER2 antibody(such as Herceptin®) and anti-VEGF antibody (such as Avastin®)). In somevariations, there is provided a method of treating a proliferativedisease (such as cancer) in an individual, comprising administering tothe individual a) an effective amount of a composition comprisingnanoparticles comprising paclitaxel and an albumin (such as Abraxane®),b) an effective amount of a platinum-based agent (such as carboplatin),and c) a therapeutic antibody (such as ant-HER2 antibody (such asHerceptin®) and anti-VEGF antibody (such as Avastin®)). In somevariations, the method is for treatment of any of advanced breastcancer, metastatic breast cancer, breast cancer in an adjuvant setting,and lung cancer (including NSCLC and advanced NSCLC). In somevariations, there is provided a method of treating metastatic cancer inan individual, comprising administering to the individual 75 mg/m²paclitaxel/albumin nanoparticle composition (such as Abraxane®) andcarboplatin, AUC=2, wherein the administration is carried out weekly forthree weeks with the fourth week off. In some variations, the methodfurther comprises weekly administering about 2-4 mg/kg of Herceptin®

In some variations, there is provided a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), b) an effective amount of a platinum-based agent (such ascarboplatin), and c) a vinca alkaloid (such as Navelbine®). In somevariations, there is provided a method of treating a proliferativedisease (such as cancer) in an individual, comprising administering tothe individual a) an effective amount of a composition comprisingnanoparticles comprising paclitaxel and an albumin (such as Abraxane®),b) an effective amount of a platinum-based agent (such as carboplatin),and c) a vinca alkaloid (such as Navelbine®). In some variations, themethod is for treatment of lung cancer.

In some variations, the invention provides a method of treating aproliferative disease (such as cancer) in an individual, comprisingadministering to the individual a) an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin), b) an effective amount of an alkylating agent (such ascyclophosphamide) and c) an anthracycline antibiotic (such asadriamycin). In some variations, the invention provides a method oftreating a proliferative disease (such as cancer) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising paclitaxel and analbumin, b) an effective amount of an alkylating agent (such ascyclophosphamide) and c) an anthracycline antibiotic (such asadriamycin). In some variations, the method is for treatment of an earlystage breast cancer. In some variations, the method is for treatment ofa breast cancer in an adjuvant or a neoadjuvant setting. For example, insome variations, there is provided a method of treating an early stagebreast cancer in an individual, comprising administering 260 mg/m²paclitaxel/albumin nanoparticle composition (such as Abraxane®), 60mg/m² adriamycin, and 600 mg/m² cyclophosphamide, wherein theadministration is carried out once every two weeks.

Other variations are provided in Table 1. For example, in somevariations, there is provided a method of treating advanced breastcancer in an individual, comprising administering to the individual a)an effective amount of a composition comprising nanoparticles comprisinga paclitaxel and an albumin (such as Abraxane®), b) an effective amountof carboplatin. In some variations, the method further comprisesadministering an effective amount of Herceptin® to the individual. Insome variations, there is provided a method of treating metastaticbreast cancer in an individual, comprising administering to theindividual a) an effective amount of a composition comprisingnanoparticles comprising paclitaxel and an albumin (such as Abraxane®),b) an effective amount of gemcitabine. In some variations, there isprovided a method of treating advanced non-small cell lung cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising paclitaxeland an albumin (such as Abraxane®), b) an effective amount ofcarboplatin.

In some variations, there is provided a composition comprisingnanoparticles comprising a taxane (such as paclitaxel, docetaxel, orortataxel) and a carrier protein (such as albumin) and at least oneother chemotherapeutic agent. The compositions described herein maycomprise effective amounts of the taxane and the chemotherapeutic agentfor the treatment of a proliferative disease (such as cancer). In somevariations, the chemotherapeutic agent and the taxane are present in thecomposition at a predetermined ratio, such as the weight ratiosdescribed herein. In some variations, the invention provides asynergistic composition of an effective amount of a compositioncomprising nanoparticles comprising a taxane (such as paclitaxel,docetaxel, or ortataxel) and an effective amount of at least one otherchemotherapeutic agent. In some variations, the other chemotherapeuticagent is an anti-VEGF antibody (such as bevacizumab, for example,Avastin®).

In some variations, the invention provides pharmaceutical compositionscomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin) for use in the treatment of a proliferative disease (such ascancer), wherein said use comprises simultaneous and/or sequentialadministration of at least one other chemotherapeutic agent. In somevariations, the invention provides a pharmaceutical compositioncomprising a chemotherapeutic agent for use in the treatment of aproliferative disease (such as cancer), wherein said use comprisessimultaneous and/or sequential administration of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin). In some variations, the invention providestaxane-containing nanoparticle compositions and compositions comprisingone other chemotherapeutic agent for simultaneous, and/or sequential usefor treatment of a proliferative disease (such as cancer).

Modes of Administration

The composition comprising nanoparticles comprising taxane (alsoreferred to as “nanoparticle composition”) and the chemotherapeuticagent can be administered simultaneously (i.e., simultaneousadministration) and/or sequentially (i.e., sequential administration).

In some variations, the nanoparticle composition and thechemotherapeutic agent (including the specific chemotherapeutic agentsdescribed herein) are administered simultaneously. The term“simultaneous administration,” as used herein, means that thenanoparticle composition and the chemotherapeutic agent are administeredwith a time separation of no more than about 15 minute(s), such as nomore than about any of 10, 5, or 1 minutes. When the drugs areadministered simultaneously, the drug in the nanoparticles and thechemotherapeutic agent may be contained in the same composition (e.g., acomposition comprising both the nanoparticles and the chemotherapeuticagent) or in separate compositions (e.g., the nanoparticles arecontained in one composition and the chemotherapeutic agent is containedin another composition). For example, the taxane and thechemotherapeutic agent may be present in a single composition containingat least two different nanoparticles, wherein some of the nanoparticlesin the composition comprise the taxane and a carrier protein, and someof the other nanoparticles in the composition comprise thechemotherapeutic agent and a carrier protein. The invention contemplatesand encompasses such compositions. In some variations, only the taxaneis contained in nanoparticles. In some variations, simultaneousadministration of the drug in the nanoparticle composition and thechemotherapeutic agent can be combined with supplemental doses of thetaxane and/or the chemotherapeutic agent.

In some variations, the nanoparticle composition and thechemotherapeutic agent are administered sequentially. The term“sequential administration” as used herein means that the drug in thenanoparticle composition and the chemotherapeutic agent are administeredwith a time separation of more than about 15 minutes, such as more thanabout any of 20, 30, 40, 50, 60 or more minutes. Either the nanoparticlecomposition or the chemotherapeutic agent may be administered first. Thenanoparticle composition and the chemotherapeutic agent are contained inseparate compositions, which may be contained in the same or differentpackages.

In some variations, the administration of the nanoparticle compositionand the chemotherapeutic agent are concurrent, i.e., the administrationperiod of the nanoparticle composition and that of the chemotherapeuticagent overlap with each other. In some variations, the administration ofthe nanoparticle composition and the chemotherapeutic agent arenon-concurrent. For example, in some variations, the administration ofthe nanoparticle composition is terminated before the chemotherapeuticagent is administered. In some variations, the administration of thechemotherapeutic agent is terminated before the nanoparticle compositionis administered. The time period between these two non-concurrentadministrations can range from about two to eight weeks, such as aboutfour weeks.

The dosing frequency of the drug-containing nanoparticle composition andthe chemotherapeutic agent may be adjusted over the course of thetreatment, based on the judgment of the administering physician. Whenadministered separately, the drug-containing nanoparticle compositionand the chemotherapeutic agent can be administered at different dosingfrequency or intervals. For example, the drug-containing nanoparticlecomposition can be administered weekly, while a chemotherapeutic agentcan be administered more or less frequently. In some variations,sustained continuous release formulation of the drug-containingnanoparticle and/or chemotherapeutic agent may be used. Variousformulations and devices for achieving sustained release are known inthe art.

The nanoparticle composition and the chemotherapeutic agent can beadministered using the same route of administration or different routesof administration. In some variations (for both simultaneous andsequential administrations), the taxane in the nanoparticle compositionand the chemotherapeutic agent are administered at a predeterminedratio. For example, in some variations, the ratio by weight of thetaxane in the nanoparticle composition and the chemotherapeutic agent isabout 1 to 1. In some variations, the weight ratio may be between about0.001 to about 1 and about 1000 to about 1, or between about 0.01 toabout 1 and 100 to about 1. In some variations, the ratio by weight ofthe taxane in the nanoparticle composition and the chemotherapeuticagent is less than about any of 100:1, 50:1, 30:1, 10:1, 9:1, 8:1, 7:1,6:1, 5:1, 4:1, 3:1, 2:1, and 1:1 In some variations, the ratio by weightof the taxane in the nanoparticle composition and the chemotherapeuticagent is more than about any of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 30:1, 50:1, 100:1. Other ratios are contemplated.

The doses required for the taxane and/or the chemotherapeutic agent may(but not necessarily) be lower than what is normally required when eachagent is administered alone. Thus, in some variations, a subtherapeuticamount of the drug in the nanoparticle composition and/or thechemotherapeutic agent are administered. “Subtherapeutic amount” or“subtherapeutic level” refer to an amount that is less than thetherapeutic amount, that is, less than the amount normally used when thedrug in the nanoparticle composition and/or the chemotherapeutic agentare administered alone. The reduction may be reflected in terms of theamount administered at a given administration and/or the amountadministered over a given period of time (reduced frequency).

In some variations, enough chemotherapeutic agent is administered so asto allow reduction of the normal dose of the drug in the nanoparticlecomposition required to effect the same degree of treatment by at leastabout any of 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or more. Insome variations, enough drug in the nanoparticle composition isadministered so as to allow reduction of the normal dose of thechemotherapeutic agent required to affect the same degree of treatmentby at least about any of 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, ormore.

In some variations, the dose of both the taxane in the nanoparticlecomposition and the chemotherapeutic agent are reduced as compared tothe corresponding normal dose of each when administered alone. In somevariations, both the taxane in the nanoparticle composition and thechemotherapeutic agent are administered at a subtherapeutic, i.e.,reduced, level. In some variations, the dose of the nanoparticlecomposition and/or the chemotherapeutic agent is substantially less thanthe established maximum toxic dose (MTD). For example, the dose of thenanoparticle composition and/or the chemotherapeutic agent is less thanabout 50%, 40%, 30%, 20%, or 10% of the MTD.

A combination of the administration configurations described herein canbe used. The combination therapy methods described herein may beperformed alone or in conjunction with another therapy, such as surgery,radiation, chemotherapy, immunotherapy, gene therapy, and the like.Additionally, a person having a greater risk of developing theproliferative disease may receive treatments to inhibit or and/or delaythe development of the disease.

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be approximately thosealready employed in clinical therapies wherein the chemotherapeuticagent are administered alone or in combination with otherchemotherapeutic agents. Variation in dosage will likely occur dependingon the condition being treated. As described above, in some variations,the chemotherapeutic agents may be administered at a reduced level.

The nanoparticle compositions described herein can be administered to anindividual (such as human) via various routes, such as parenterally,including intravenous, intra-arterial, intraperitoneal, intrapulmonary,oral, inhalation, intravesicular, intramuscular, intra-tracheal,subcutaneous, intraocular, intrathecal, or transdermal. For example, thenanoparticle composition can be administered by inhalation to treatconditions of the respiratory tract. The composition can be used totreat respiratory conditions such as pulmonary fibrosis, broncheolitisobliterans, lung cancer, bronchoalveolar carcinoma, and the like. Insome variations, the nanoparticle composition is administratedintravenously. In some variations, the nanoparticle composition isadministered orally.

The dosing frequency of the administration of the nanoparticlecomposition depends on the nature of the combination therapy and theparticular disease being treated. An exemplary dosing frequency include,but is not limited to, weekly without break; weekly, three out of fourweeks; once every three weeks; once every two weeks; weekly, two out ofthree weeks. See also Table 1.

The dose of the taxane in the nanoparticle composition will vary withthe nature of the combination therapy and the particular disease beingtreated. The dose should be sufficient to effect a desirable response,such as a therapeutic or prophylactic response against a particulardisease. An exemplary dose of the taxane (in some variations paclitaxel)in the nanoparticle composition include, but is not limited to, aboutany of 50 mg/m², 60 mg/m², 75 mg/m², 80 mg/m², 90 mg/m², 100 mg/m², 120mg/m², 160 mg/m², 175 mg/m², 200 mg/m², 210 mg/m², 220 mg/m², 260 mg/m²,and 300 mg/m². For example, the dosage of paclitaxel in a nanoparticlecomposition can be in the range of 100-400 mg/m² when given on a 3 weekschedule, or 50-250 mg/m² when given on a weekly schedule. See alsoTable 1.

Other exemplary dosing schedules for the administration of thenanoparticle composition (such as paclitaxel/albumin nanoparticlecomposition, for example Abraxane®) include, but are not limited to, 100mg/m², weekly, without break; 75 mg/m² weekly, 3 out of four weeks; 100mg/m², weekly, 3 out of 4 weeks; 125 mg/m², weekly, 3 out of 4 weeks;125 mg/m², weekly, 2 out of 3 weeks; 130 mg/m², weekly, without break;175 mg/m², once every 2 weeks; 260 mg/m², once every 2 weeks; 260 mg/m²,once every 3 weeks; 180-300 mg/m², every three weeks; 60-175 mg/m²,weekly, without break. In addition, the taxane (alone or in combinationtherapy) can be administered by following a metronomic dosing regimedescribed herein.

Exemplary dosing regimes for the combination therapy of nanoparticlecomposition (such as paclitaxel/albumin nanoparticle composition, forexample Abraxane®) and other agents include, but are not limited to, 125mg/m² weekly, two out of three weeks, plus 825 mg/m² Xeloda®, daily; 260mg/m² once every two weeks, plus 60 mg/m² adriamycin and 600 mg/m²cyclophosphamide, once every two weeks; 220-340 mg/m² once every threeweeks, plus carboplatin, AUC=6, once every three weeks; 100-150 mg/m²weekly, plus carboplatin, AUC=6, once every three weeks; 175 mg/m² onceevery two weeks, plus 2000 mg/m² gemcitabine and 50 mg/m² epirubicin,once every two weeks; and 75 mg/m² weekly, three out of four weeks, pluscarboplatin, AUC=2, weekly, three out of four weeks.

In some variations, the nanoparticle composition of the taxane and thechemotherapeutic agent is administered according to any of the dosingregimes described in Table 1.

In some variations, there is provided a method of treating breast cancerin an individual comprising administering to the individual: a) aneffective amount of a composition comprising nanoparticles comprising ataxane (such as paclitaxel) and an albumin, and b) an effective amountof at least one other chemotherapeutic agent as provided in Rows 1 to 35in Table 1. In some variations, the administration of the nanoparticlecomposition and the chemotherapeutic agent may be any of the dosingregimes as indicated in Rows 1 to 35 in Table 1. In some variations,there is provided a method of treating metastatic breast cancer in anindividual comprising administering to the individual: a) an effectiveamount of a composition comprising nanoparticles comprising a taxane(such as paclitaxel) and an albumin, and b) an effective amount of atleast one other chemotherapeutic agent as provided in Rows 2, 4-8, and10-15 in Table 1. In some variations, the administration of thenanoparticle composition and the chemotherapeutic agent may be any ofthe dosing regimes as indicated in Rows 2, 4-8, and 10-15 in Table 1.

In some variations, there is provided a method of treating advancedbreast cancer in an individual comprising administering to theindividual: a) an effective amount of a composition comprisingnanoparticles comprising a taxane (such as paclitaxel) and an albumin,and b) an effective amount of at least one other chemotherapeutic agentas provided in Rows 1 and 16 in Table 1. In some variations, theadministration of the nanoparticle composition and the chemotherapeuticagent may be any of the dosing regimes as indicated in Rows 1 and 16 inTable 1. In some variations, there is provided a method of treatingstage IV breast cancer in an individual comprising administering to theindividual: a) an effective amount of a composition comprisingnanoparticles comprising a taxane (such as paclitaxel) and an albumin,and b) an effective amount of at least one other chemotherapeutic agentas provided in Row 3 in Table 1. In some variations, the administrationof the nanoparticle composition and the chemotherapeutic agent may bethe dosing regime as indicated in Row 3 in Table 1.

In some variations, there is provided a method of treating breast cancerin an individual in an adjuvant setting comprising administering to theindividual: a) an effective amount of a composition comprisingnanoparticles comprising a taxane (such as paclitaxel) and an albumin,and b) an effective amount of at least one other chemotherapeutic agentas provided in Rows 18 to 24 in Table 1. In some variations, theadministration of the nanoparticle composition and the chemotherapeuticagent may be any of the dosing regimes as indicated in Rows 18 to 24 inTable 1.

In some variations, there is provided a method of treating breast cancerin an individual in a neoadjuvant setting comprising administering tothe individual: a) an effective amount of a composition comprisingnanoparticles comprising a taxane (such as paclitaxel) and an albumin,and b) an effective amount of at least one other chemotherapeutic agentas provided in Rows 25 to 35 in Table 1. In some variations, theadministration of the nanoparticle composition and the chemotherapeuticagent may be any of the dosing regimes as indicated in Rows 25 to 35 inTable 1.

In some variations, there is provided a method of treating lung cancerin an individual comprising administering to the individual: a) aneffective amount of a composition comprising nanoparticles comprising ataxane (such as paclitaxel) and an albumin, and b) an effective amountof at least one other chemotherapeutic agent as provided in Rows 36 to48 in Table 1. In some variations, the administration of thenanoparticle composition and the chemotherapeutic agent may be any ofthe dosing regimes as indicated in Rows 36 to 48 in Table 1.

In some variations, there is provided a method of treating NSCLC(including advanced NSCLC and first line NSCLC) in an individualcomprising administering to the individual: a) an effective amount of acomposition comprising nanoparticles comprising a taxane (such aspaclitaxel) and an albumin, and b) an effective amount of at least oneother chemotherapeutic agent as provided in Rows 36-40 and 42-43 inTable 1. In some variations, the administration of the nanoparticlecomposition and the chemotherapeutic agent may be any of the dosingregimes as indicated in Rows 36-40 and 42-43 in Table 1. In somevariations, there is provided a method of treating advanced solid tumormalignancy in the lung in an individual comprising administering to theindividual: a) an effective amount of a composition comprisingnanoparticles comprising a taxane (such as paclitaxel) and an albumin,and b) an effective amount of at least one other chemotherapeutic agentas provided in Row 41 in Table 1. In some variations, the administrationof the nanoparticle composition and the chemotherapeutic agent may bethe dosing regimes as indicated in Row 41 in Table 1. In somevariations, there is provided a method of treating SCLC in an individualcomprising administering to the individual: a) an effective amount of acomposition comprising nanoparticles comprising a taxane (such aspaclitaxel) and an albumin, and b) an effective amount of at least oneother chemotherapeutic agent as provided in Row 48 in Table 1. In somevariations, the administration of the nanoparticle composition and thechemotherapeutic agent may be the dosing regimes as indicated in Row 48in Table 1.

In some variations, there is provided a method of treating ovariancancer in an individual comprising administering to the individual: a)an effective amount of a composition comprising nanoparticles comprisinga taxane (such as paclitaxel) and an albumin, and b) an effective amountof at least one other chemotherapeutic agent as provided in Rows 49 to52 in Table 1. In some variations, the administration of thenanoparticle composition and the chemotherapeutic agent may be any ofthe dosing regimes as indicated in Rows 49 to 52 in Table 1.

In some variations, there is provided a method of treating head and neckcancer in an individual comprising administering to the individual: a)an effective amount of a composition comprising nanoparticles comprisinga taxane (such as paclitaxel) and an albumin, and b) an effective amountof at least one other chemotherapeutic agent as provided in Rows 53 to55 in Table 1. In some variations, the administration of thenanoparticle composition and the chemotherapeutic agent may be any ofthe dosing regimes as indicated in Rows 53 to 55 in Table 1.

In some variations, there is provided a method of treating solid tumor(including advanced solid tumor) in an individual comprisingadministering to the individual: a) an effective amount of a compositioncomprising nanoparticles comprising a taxane (such as paclitaxel) and analbumin, and b) an effective amount of at least one otherchemotherapeutic agent as provided in Rows 56 to 59 in Table 1. In somevariations, the administration of the nanoparticle composition and thechemotherapeutic agent may be any of the dosing regimes as indicated inRows 56 to 59 in Table 1.

In some variations, there is provided a method of treating melanoma(including metastatic melanoma) in an individual comprisingadministering to the individual: a) an effective amount of a compositioncomprising nanoparticles comprising a taxane (such as paclitaxel) and analbumin, and b) an effective amount of at least one otherchemotherapeutic agent as provided in Rows 60-63 in Table 1. In somevariations, the administration of the nanoparticle composition and thechemotherapeutic agent may be any of the dosing regimes as indicated inRows 60 to 63 in Table 1.

In some variations, there is provided a method of treating metastaticcolorectal cancer in an individual comprising administering to theindividual: a) an effective amount of a composition comprisingnanoparticles comprising a taxane (such as paclitaxel) and an albumin,and b) an effective amount of at least one other chemotherapeutic agentas provided in Row 64 in Table 1. In some variations, the administrationof the nanoparticle composition and the chemotherapeutic agent may bethe dosing regime as indicated in Row 64 in Table 1.

In some variations, there is provided a method of treating pancreaticcancer in an individual comprising administering to the individual: a)an effective amount of a composition comprising nanoparticles comprisinga taxane (such as paclitaxel) and an albumin, and b) an effective amountof at least one other chemotherapeutic agent as provided in Rows 65 to66 in Table 1. In some variations, the administration of thenanoparticle composition and the chemotherapeutic agent may be any ofthe dosing regimes as indicated in Rows 65 to 66 in Table 1.

TABLE 1 Row Study therapy No. Combination Regime/Dosage type Protocoltitle 1. ABX + ABX: 100 mg/m² D1, 8, 15 Advanced A phase II study ofCarboplatin + q4wk × 6 HER2+ Breast weekly dose-dense Herceptin ® Carbo:AUC = 2 D1, 8, 15 q4wk × 6 Cancer nanoparticle paclitaxel Herceptin ®: 4mg/kg on wk 1, 2 mg/kg (ABI-007) all subsequent weeks carboplatin ™,with Herceptin ® as first or second-line therapy of advanced HER2+breast cancer 2. ABX alone ABX: 125 mg/m² Metastatic Phase II trial ofweekly (+Herceptin ®) qwk × 3/4 Breast Cancer Abraxane ® monotherapy for1st- line MBC (plus Herceptin ® in HER2+ pts) 3. ABX + L1: ABX: 80 mg/mStage IV Phase I-II study weekly Navelbine ® Nav: 15 mg/m² Breast CancerABX + Navelbine ®, (±G-CSF) L2: ABX: 90 mg/m² with or without G-CSF,Nav: 20 mg/m² in stage IV breast L3: ABX: 100 mg/m² cancer Nav: 22.5mg/m² L4: ABX: 110 mg/m² Nav: 25 mg/m² L5: ABX: 125 mg/m² Nav: 25 mg/m²qwk all levels 4. ABX + Xeloda ® ABX: 125 mg/m² qwk × 2/3 MetastaticPhase II 1st-line ABX + Xeloda ®: 825 mg/m² D1-14 Breast Cancer Xeloda ®MBC trial q3wk 5. ABX + Metastatic Phase I/II trial ABX AnthracyclineBreast Cancer plus Doxil ® for MBC plus limited PK 6. ABX + ABX: 125mg/m² Metastatic Randomized Phase II Gemcitabine Gem: 1000 mg/m² BreastCancer Trial of Weekly nab qwk × 2/3 (nanoparticle albuminbound)-Paclitaxel (nab- paclitaxel) in Combination with Gemcitabine inPatients with HER2 Negative Metastatic Breast Cancer 7. ABX + MetastaticPhase I/II Abraxane ® + Lapatinib Breast Cancer GW572016 8. ABX + ABX:100 mg/m² qwk × 3/4 Metastatic Phase I dose escalation LapatinibLapatinib: starting at 1000 mg/d × Breast Cancer study of a 2 day oral 2days Lapatinib chemosensitization pulse given prior to weeklyintravenous Abraxane ® in patients with advanced solid tumors 9. ABX +FEC ABX: 220 mg/m² q2wk × 6 Breast Cancer Phase II preoperative(+Herceptin ®) followed by trial of Abraxane ® FEC: 4 cycles(+Herceptin ® for followed by FEC HER2+ pts) (+Herceptin ® asappropriate) in breast cancer 10. ABX + ABX: 100 mg/m² qwk D1, 8, 15Metastatic Phase II safety and Carboplatin + Carbo: AUC = 2 qwk D1, 8,15 Breast Cancer tolerability study of Avastin ® Avastin ®: 10 mg/m²q2wk (HER2−, ER−, Abraxane ®, Avastin ® PR−) and carboplatin in triplenegative metastatic breast cancer patients 11. ABX + Avastin ® ABX: 130mg/m² qwk + Metastatic Three arm phase II trial Avastin ® Breast Cancerin 1^(st) line HER2− vs negative MBC patients ABX: 260 mg/m² q2wk +Avastin ® vs ABX: 260 mg/m² q3wk + Avastin ® 12. ABX + Avastin ® ABX:125 mg/m² qwk × 3/4 + Metastatic Single arm study of Avastin ® BreastCancer Abraxane ® and Avastin ® in 1^(st) line MBS 13. ABX + Avastin ®ABX + Avastin ® qwk Metastatic Randomized Phase III vs Breast Cancertrial in 1^(st) line and 2^(nd) Taxol ® + Avastin ® qwk line MBC withbiological correlates analysis 14. ABX + Xeloda ® + Metastatic Phase IIAbraxane ® in Lapatinib Breast Cancer combination with Xeloda ® andLapatinib for metastatic breast cancer 15. ABX + ABX: 3000 mg/m² D1 q3wkMetastatic Single arm Phase II Gemcitabine Gem: 1250 mg/m² D1, 8 q3wkBreast Cancer study of Abraxane ® and gemcitabine for 1^(st) line MBC16. ABX + RAD001 Advanced Phase I/II study of Breast Cancer Abraxane ®in combination with RAD001 in patients with advanced breast cancer 17.ABX + Sutent ® Breast Cancer Phase I study of Abraxane ® in combinationwith Sutent ® 18. ABX + AC + G- AC + G-CSF q2wk × 4 Breast Cancer-Abraxane ® in dose- (+CSF followed by Adjuvant dense adjuvantHerceptin ®) ABX: 260 mg/m² q2wk × 4 chemotherapy for early(+Herceptin ® for HER2+ pts) stage breast cancer 19. ABX + AC + G- Dosedense AC + G-CSF Breast Cancer- Phase II pilot adjuvant CSF followed byABX Adjuvant trial of Abraxane ® in (+Herceptin ®) (+Herceptin ® forHER2+ pts) breast cancer qwk 20. ABX + AC AC followed by ABX: 260 mg/m²Breast Cancer- Adjuvant Dose dense vs Adjuvant Registrational Trial ACfollowed by Taxol ® Rx length 16 wks 21. ABX + AC AC q2wk followed byBreast Cancer- Phase II dose dense (+G-CSF) ABX: 260 mg/m² + G-CSF q2wkAdjuvant pilot adjuvant study of Rx length 16 wks Abraxane ® in breastcancer 22. ABX + AC Dose dense AC followed by Breast Cancer- Pilotadjuvant breast (+Avastin ®) ABX (+Avastin ® in HER2+ pts) Adjuvantcancer study 23. ABX + AC AC Breast Cancer- BIG study: Dose densefollowed by ABX Adjuvant vs standard adjuvant q2wk or q3wk chemotherapy24. ABX (ABI-007) + AC followed by Breast Cancer- Phase II - Pilot StudyAC + ABX q2wk × 4 Adjuvant Evaluating the Safety of Neulasta ® aDose-Dense Regime - AC × 4 => ABI-007 × 4 Q 2 WEEKS + Neulasta ® - Givenas Adjuvant Chemotherapy of High- Risk Women with Early Breast Cancer25. ABX + FEC ABX: 100 mg/m² qwk × 12 Locally A Phase II Study of(+Herceptin ®) followed by Advanced Breast Neoadjuvant 5-FU: 500 mg/m²q3wk Cancer - Chemotherapy with Epirubicin: 100 mg/m² NeoadjuvantSequential Weekly (without Herceptin ®) Nanoparticle Albumin or BoundPaclitaxel Epirubicin: 75 mg/m² (Abraxane ®) Followed (with Herceptin ®for HER2+ by 5-Fluorouracil, pts) Epirubicin, Cyclophosphamide: 500mg/m² Cyclophosphamide q3wk (FEC) in Locally Advanced Breast Cancer 26.ABX + Arm 1: Neoadjuvant: Gem: 2000 mg/m², Breast Cancer- Phase II Trialof Dose Gemcitabine + ABX: 175 mg/m², Epi Neoadjuvant Dense NeoadjuvantEpirubicin 50 mg/m² Gemcitabine, q2wk × 6 Epirubicin, ABI-007 Arm 2:Adjuvant: Gem: 2000 mg/m², (GEA) in Locally ABX: 220 mg/m² Advanced orq2wk × 4 Inflammatory Breast Cance 27. ABX + ABX: 260 mg/m² q2wk +Breast Cancer- Phase II Multi-center Herceptin ® Herceptin ® Neoadjuvantstudy neoadjuvant. followed by Navelbine ® + Herceptin ® 28. ABX + TACBreast Cancer- 3 arms Randomized Carboplatin vs Neoadjuvant dose densephase II (+Herceptin ®) + AC followed by ABX + carbo trial ofneoadjuvant AC vs chemotherapy in AC followed by ABX + carbo + patientswith breast Herceptin ® cancer 29. ABX + ABX: 260 mg/m² q3wk × 4 BreastCancer- Phase II neoadjuvant Capecitabine Xeloda ® 850 mg/m² D1-14Neoadjuvant trial of Abraxane ® and q3wk × 4 capecitabine in locallyadvanced breast cancer 30. ABX + ABX qwk Breast Cancer- Phase I/II trialof Carboplatin carbo qwk + Neoadjuvant neoadjuvant (+Avastin ®)Avastin ® in HER2+ pts chemotherapy (NCT) with weekly nanoparticlepaclitaxel (ABI-007, Abraxane ®) in combination with carboplatin andAvastin ® in clinical stage I-III. 31. ABX + ABX: 100 mg/m² qwk × 3/4Breast Cancer- Phase II study of Carboplatin + Carbo: AUC = 5 +Neoadjuvant weekly bevacizumab Herceptin ® + Herceptin ® + administeredwith Avastin ® Avastin ® weekly trastuzumab, 4 week cycle × 6 ABI-007,and carboplatin as preoperative therapy in HER2-neu gene amplifiedbreast cancer tumors 32. ABX + ABX: 260 mg/m² q3wk Breast Cancer- Pilotneoadjuvant trial Lapatinib Lapatinib: 1000 mg/day Neoadjuvant withcombination of ABI-007 (Abraxane ®) and GW572016 (Lapatinib) 33. ABX +ABX: 200 mg/m² Breast Cancer- Phase II neoadjuvant Capecitabine q3wk × 4Neoadjuvant trial of Abraxane ® and Xeloda ®: 1000 mg/m² capecitabine inlocally D1-14 q3wk × 4 advanced breast cancer 34. ABX ± Avastin ® + ABXqwk ± Avastin ® followed Breast Cancer- Phase III trial of AC by A qwk +C daily Neoadjuvant paclitaxel vs Abraxane ® (+G-CSF) vs with or withoutTaxol ® qwk ± Avastin ® followed Avastin ® in by A qwk + C dailycombination with doxorubicin and cyclophosphamide plus G-CSF 35. ABX +AC ABX followed by AC Breast Cancer- Phase II neoadjuvant Neoadjuvanttrial with gene expression analyses 36. ABX + ABX: 300 mg/m² q3wk 1^(st)line An open label phase II Carboplatin + Carbo: AUC = 6 q3wk Advancedtrial of Abraxane ®, Avastin ® Avastin ®: 15 mg/kg NSCLC carboplatin and4 cycles Avastin ® in patients with advanced non- squamous non-smallcell lung cancer 37. ABX + L1: ABX: 225 mg/m² Advanced Phase II toxicitypilot Carboplatin L2: ABX: 260 mg/m² NSCLC study of Abraxane ® and L3:ABX: 300 mg/m² carboplatin in advanced Cohorts 1-4: ABX q3wk non-smallcell lung Cohorts 5-7: ABX weekly cancer. Cohort 8: 75 additionalpatients Carbo fixed at AUC = 6 q3wk 38. ABX + Carbo: AUC = 6 + ABX1^(st) line NSCLC Phase III Registration - Carboplatin vs NSCLC 1st linetherapy Carbo: AUC = 6 + Taxol ®: 225 mg/m² 39. ABX + ABX: 100 mg/m² d1,8, 15 1^(st) line NSCLC Phase II Trial of weekly Carboplatin Carbo: AUC= 6 q4wk Abraxane ® plus Amendment: ABX: 125 mg/m² carboplatin in1st-line D1, 8, 15 NSCLC 40. ABX + Weekly NSCLC Carboplatin + Avastin ®41. ABX + Arm 1: ABX: 100, 125, 150 mg/m² Lung Cancer - Phase I Trial ofCarboplatin D1, 8, 15 q4wk Advanced Solid carboplatin and Arm 2: ABX220, 260, 300, 340 mg/m² Tumor Abraxane ® on a weekly q3wk Malignancyand every three week Arm 3: ABX 100, 125, 150 mg/m² schedule in patientsD1, 8 with Advanced Solid Carbo: AUC = 6 in all arms Tumor Malignancies42. ABX + NSCLC Abraxane ® in Gemcitabine or combination with ABX +Avastin ® gemcitabine or Avastin ® 43. ABX + NSCLC Phase I trial ofGemcitabine Abraxane ® in combination with gemcitabine 44. ABX + ABX:225, 260, 300 mg/m² Lung Cancer Phase I/II study of Carboplatin + Carbo:AUC = 6 Abraxane ® and Avastin ® q3wk + carboplatin AUC 6, Avastin ®plus Avastin ® (Standard 3 + 3 Phase I design; PhII: 40 pts) 45. ABX +Alimta ® ABX: 220, 260, 300 mg/m² Lung Cancer Phase I/II study of q3wkAbraxane ® + Alimta ® Pemtrexed: 500 mg q3wk for 2nd-line NSCLC 46.ABX + Cisplatin Lung Cancer Phase I/II trial of Abraxane ® pluscisplatin in advanced NSCLC 47. ABX + Lung Cancer Phase I/II study ofNavelbine ® + Abraxane ®, Cisplatin Navelbine ®, and Cisplatin fortreatment of advanced NSCLC 48. ABX + ABX: 300 mg/m² q3wk SCLC Phase IItrial of Carboplatin Carbo: AUC = 6 q3wk Abraxane ® and carboplatin inextensive stage small cell lung cancer 49. ABX + ABX: 100 mg/m² qwk ×3/4 Ovarian Cancer A phase II trial of Carboplatin Carbo: AUC = 6Abraxane ® + Carboplatin in recurrent ovarian cancer 50. ABX + ABX: qwkOvarian Cancer Phase I study of Carboplatin ABX: q3w Abraxane ® pluscarbo Carbo: AUC = 6 both arms for treatment of advanced ovarian cancer51. ABX + ABX: TBD by ABI-CA034 Ovarian Cancer 1st line, optimallyCarboplatin vs debulked, registration Taxol ® 175 mg/m² trial. Carbo AUC6 + Carbo: AUC = 6 in both arms ABX vs Carbo + Taxol ® 175 mg/m².Endpoint: relapse free survival, survival 52. ABX + Avastin ® ABX: 100mg/m² qwk × 3/4 Ovarian Cancer Phase II study of Avastin ®: 10 mg/m²q2wk bevacizumab with Abraxane ® in patients with recurrent, platinumresistant primary epithelial ovarian or primary peritoneal carcinoma 53.ABX + 5-FU + ABX: D1 Head and Neck Unresectable localized Cisplatin5-FU: 750 mg/m² CIV × 5 Cancer head and neck cancer cisplatin: 75 mg/m²D1 Phase II Abraxane ® in followed by XRT/surgery combination with 5-FUand cisplatin 54. ABX + 5-FU + 5-FU: 750 mg/m² CIV × 5 Head and NeckUnresectable localized Cisplatin cisplatin: 75 mg/m² D1 ± Cancer headand neck cancer ABX D1 Phase III 5-FU and followed by XRT/surgerycisplatin with or without Abraxane ® 55. ABX + Head and Neck Phase IImulticenter trial Cetuximab Cancer of Abraxane ® in combination withcetuximab in 1^(st) line treatment of locally advanced or metastatichead and neck cancer 56. ABX + ABX: 100 mg/m² qwk Solid Tumors Phase IStudy of Rapamycin Rapamycin: 5-40 mg dose Rapamycin in escalationCombination with Abraxane ® in Advanced Solid Tumors 57. ABX + SolidTumors Phase I trial of Abraxane ® Satraplatin and Satraplatin 58. ABX +ABX: 180, 220, 260, 300, 340 mg/m² Advanced Solid Phase I Trial ofAbraxane ® Gemcitabine q3wk Tumors in combination with Gemcitabine: 1000mg/m² D1 Gemcitabine and D8 59. ABX + Gefitinib ABX: 100 mg/m² qwk × 3/4Advanced Solid Phase I dose escalation Gefitinib starting at 1000 mg/d ×2 Tumors study of gefitinib chemosensitization pulse given prior toweekly Abraxane ® 60. ABX + Avastin ® Metastatic Phase II study ofMelanoma Abraxane ® and Avastin ® in metastatic melanoma 61. ABX +Avastin ® Melanoma Abraxane ® and Avastin ® as therapy for patients withmalignant melanoma 62. ABX + Metastatic Phase II study of CarboplatinMelanoma Abraxane ® and carboplatin in metastatic melanoma 63. ABX +ABX: qwk Metastatic Phase II study of Sorafenib + Sorafenib: D2-19Melanoma Abraxane ® in Carboplatin Carbo: AUC = 6 D1 combination withcarboplatin and sorafenib in metastatic melanoma 64. ABX + MetastaticPhase II trial of Capecitabine Colorectal Abraxane ® in Cancer (aftercombination with failure of Xeloda ® for previously oxaliplatin- treatedpatient with based therapy advance or metastatic and irinotecan-colorectal cancer based therapy) 65. ABX + Weekly Pancreatic Phase Istudy of Gemcitabine Cancer Abraxane ® in combination with gemcitabinein pancreatic cancer 66. ABX + ABX + Gem Pancreatic Phase IIIregistration trial Gemcitabine vs Cancer in pancreatic cancer Gem 67.ABX + anti- Abraxane ® combined angiogenic with anti-angiogenic agentsagents, e.g. Avastin ® 68. ABX + Abraxane ® combined proteasome withproteasome inhibitors inhibitors, e.g. Velcade ® 69. ABX + EGFRAbraxane ® combined inhibitors with EGFR inhibitors, e.g. Tarceva ®

As used in herein (for example in Table 1), ABX refers to Abraxane®;GW572016 refers to lapatinib; Xel refers to capecitabine or Xeloda®;bevacizumab is also known as Avastin®; trastuzumab is also known asHerceptin®; pemtrexed is also known as Alimta®; cetuximab is also knownas Erbitux®; gefitinib is also known as Iressa®; FEC refers to acombination of 5-fluorouracil, Epirubicin and Cyclophosphamide; ACrefers to a combination of Adriamycin plus Cyclophosphamide; TAC refersto a FDA approved adjuvant breast cancer regime; RAD001 refers to aderivative of rapamycin; NSCLC refers to non-small cell lung cancer; andSCLC refers to small cell lung cancer.

As used herein (for example in Table 1), AUC refers to area under curve;q4wk refers to a dose every 4 weeks; q3wk refers to a dose every 3weeks; q2wk refers to a dose every 2 weeks; qwk refers to a weekly dose;qwk×¾ refers to a weekly dose for 3 weeks with the 4^(th) week off;qwk×⅔ refers to a weekly dose for 2 weeks with the 3^(rd) week off.

Combination Therapy with Radiation Therapy and Surgery

In another aspect, the present invention provides a method of treatingproliferative disease (such as cancer) comprising a first therapycomprising administering a taxane (particularly nanoparticles comprisinga taxane) and a carrier protein and a second therapy comprisingradiation and/or surgery.

In some variations, the method comprises: a) a first therapy comprisingadministering to the individual a composition comprising nanoparticlescomprising an effective amount of a taxane and a carrier protein (suchas albumin) and b) a second therapy comprising radiation therapy,surgery, or combinations thereof. In some variations, the taxane iscoated with the carrier protein (such as albumin). In some variations,the second therapy is radiation therapy. In some variations, the secondtherapy is surgery.

In some variations, the method comprises a) a first therapy comprisingadministering to the individual a composition comprising nanoparticlescomprising paclitaxel and an albumin; and b) a second therapy comprisingradiation therapy, surgery, or combinations thereof. In some variations,the second therapy is radiation therapy. In some variations, the secondtherapy is surgery. In some variations, the paclitaxel/albuminnanoparticles have an average diameter of no greater than about 200 nm.In some variations, the paclitaxel/albumin nanoparticle composition issubstantially free (such as free) of surfactant (such as Cremophor). Insome variations, the weight ratio of the albumin to paclitaxel in thecomposition is about 18:1 or less, such as about 9:1 or less. In somevariations, the paclitaxel is coated with albumin. In some variations,the paclitaxel/albumin nanoparticles have an average diameter of nogreater than about 200 nm and the paclitaxel/albumin composition issubstantially free (such as free) of surfactant (such as Cremophor). Insome variations, the paclitaxel/albumin nanoparticles have an averagediameter of no greater than about 200 nm and the paclitaxel is coatedwith albumin. In some variations, the nanoparticle composition isAbraxane®.

The administration of the nanoparticle composition may be prior to theradiation and/or surgery, after the radiation and/or surgery, orconcurrent with the radiation and/or surgery. For example, theadministration of the nanoparticle composition may precede or follow theradiation and/or surgery therapy by intervals ranging from minutes toweeks. In some variations, the time period between the first and thesecond therapy is such that the taxane and the radiation/surgery wouldstill be able to exert an advantageously combined effect on the cell.For example, the taxane (such as paclitaxel) in the nanoparticlecomposition may be administered less than about any of 1, 3, 6, 9, 12,18, 24, 48, 60, 72, 84, 96, 108, 120 hours prior to the radiation and/orsurgery. In some variations, the nanoparticle composition isadministered less than about 9 hours prior to the radiation and/surgery.In some variations, the nanoparticle composition is administered lessthan about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to theradiation/surgery. In some variations, the taxane (such as paclitaxel)in the nanoparticle composition is administered less than about any of1, 3, 6, 9, 12, 18, 24, 48, 60, 72, 84, 96, 108, or 120 hours after theradiation and/or surgery. In some variations, it may be desirable toextend the time period for treatment significantly, where several daysto several weeks lapse between the two therapies.

Radiation contemplated herein includes, for example, γ-rays, X-rays(external beam), and the directed delivery of radioisotopes to tumorcells. Other forms of DNA damaging factors are also contemplated such asmicrowaves and UV irradiation are also contemplated. Radiation may begiven in a single dose or in a series of small doses in adose-fractionated schedule. The amount of radiation contemplated hereinranges from about 1 to about 100 Gy, including, for example, about 5 toabout 80, about 10 to about 50 Gy, or about 10 Gy. The total dose may beapplied in a fractioned regime. For example, the regime may comprisefractionated individual doses of 2 Gy. Dosage ranges for radioisotopesvary widely, and depends on the half-life of the isotope and thestrength and type of radiation emitted.

When the radiation comprises use of radioactive isotopes, the isotopemay be conjugated to a targeting agent, such as a therapeutic antibody,which carries the radionucleotide to the target tissue. Suitableradioactive isotopes include, but are not limited to, astatine²¹¹,¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷iron, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu,gallium⁶⁷, ³hydrogen, iodine¹²³, iodine¹³¹, indium¹¹¹, ⁵⁹ion,³²phosphorus, rhenium¹⁸⁶, ⁷⁵selenium, ³⁵sulphur, technicium^(99m),and/or yttrium⁹⁰.

In some variations, enough radiation is applied to the individual so asto allow reduction of the normal dose of the taxane (such as paclitaxel)in the nanoparticle composition required to effect the same degree oftreatment by at least about any of 5%, 10%, 20%, 30%, 50%, 60%, 70%,80%, 90%, or more. In some variations, enough taxane in the nanoparticlecomposition is administered so as to allow reduction of the normal doseof the radiation required to effect the same degree of treatment by atleast about any of 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or more.In some variations, the dose of both the taxane (such as paclitaxel) inthe nanoparticle composition and the radiation are reduced as comparedto the corresponding normal dose of each when used alone.

In some variations, the combination of administration of thenanoparticle composition and the radiation therapy producesupra-additive effect. In some variations, the taxane (such aspaclitaxel) in the nanoparticle composition is administered once at thedose of 90 mg/kg, and the radiation is applied five times at 80 Gydaily.

Surgery described herein includes resection in which all or part ofcancerous tissue is physically removed, exercised, and/or destroyed.Tumor resection refers to physical removal of at least part of a tumor.In addition to tumor resection, treatment by surgery includes lasersurgery, cryosurgery, electrosurgery, and micropically controlledsurgery (Mohs surgery). Removal of superficial surgery, precancers, ornormal tissues are also contemplated.

The radiation therapy and/or surgery may be carried out in addition tothe administration of chemotherapeutic agents. For example, theindividual may first be administered with a taxane-containingnanoparticle composition and at least one other chemotherapeutic agent,and subsequently be subject to radiation therapy and/or surgery.Alternatively, the individual may first be treated with radiationtherapy and/or surgery, which is then followed by the administration ofa nanoparticle composition and at least one other chemotherapeuticagent. Other combinations are also contemplated.

Administration of nanoparticle compositions disclosed above inconjunction with administration of chemotherapeutic agent is equallyapplicable to those in conjunction with radiation therapy and/orsurgery.

In some variations, the nanoparticle composition of the taxane and/orthe chemotherapeutic agent is administered in conjunction with radiationaccording to any of the dosing regimes described in Table 2.

In some variations, there is provided a method of treating NSCLC in anindividual comprises a) a first therapy comprising administering to theindividual a composition comprising nanoparticles comprising taxane(such as paclitaxel) and an albumin; and b) a second therapy comprisingradiation as provided in Rows 1 to 5 in Table 2. In some variations, theadministration of the nanoparticle composition and the chemotherapeuticagent may be any of the dosing regimes as indicated in Rows 1 to 5 inTable 2.

In some variations, there is provided a method of treating head and neckcancer in an individual comprises a) a first therapy comprisingadministering to the individual a composition comprising nanoparticlescomprising taxane (such as paclitaxel) and an albumin; and b) a secondtherapy comprising radiation as provided in Rows 6 to 9 in Table 2. Insome variations, the administration of the nanoparticle composition andthe chemotherapeutic agent may be any of the dosing regimes as indicatedin Rows 6 to 9 in Table 2.

In some variations, there is provided a method of treating pancreaticcancer in an individual comprises a) a first therapy comprisingadministering to the individual a composition comprising nanoparticlescomprising taxane (such as paclitaxel) and an albumin; and b) a secondtherapy comprising radiation as provided in Row 10 in Table 2. In somevariations, the administration of the nanoparticle composition and thechemotherapeutic agent may be the dosing regimes as indicated in Row 10in Table 2.

In some variations, there is provided a method of treating gastricmalignancies in an individual comprises a) a first therapy comprisingadministering to the individual a composition comprising nanoparticlescomprising taxane (such as paclitaxel) and an albumin; and b) a secondtherapy comprising radiation as provided in Row 11 in Table 2. In somevariations, the administration of the nanoparticle composition and thechemotherapeutic agent may be the dosing regimes as indicated in Row 11in Table 2.

TABLE 2 Row Study therapy No. Combination Regime/Dosage type Protocoltitle 1 ABX + NSCLC Phase I/II trial of Radiation Abraxane ® combinedwith radiation 2 ABX + NSCLC Phase I/II trial of Carboplatin +Abraxane ® and Radiation carboplatin combined with radiation. 3 ABX + 1cycle ABX/Carbo induction NSCLC Phase II chemoradiation Carboplain +followed by in NSCLC Radiation 2 or 3 times weekly pulse ABX + radiation4 ABX + NSCLC Abraxane ®/carboplatin Carboplatin + induction followed byRadiation Abraxane ® + radiation in stage III A&B PS2 NSCLC patients 5ABX + ABX qwk + carbo + radiation NSCLC Phase II study Carboplatin +followed by Radiation ABX q3wk + carbo 6 ABX + Head and Abraxane ® as aRadiation Neck Cancer radiosensitizer in head and neck cancer 7 ABX +Head and PhaseI/II Abraxane ® in Cetuximab + Neck Cancer combinationwith Radiation cetuximab and radiation 8 ABX + Induction: ABX 135 mg/m²Head and Phase I/II study of Carboplatin + qwk + carbo: AUC = 2 NeckCancer induction chemotherapy 5-FU + followed by with Abraxane ® andHydroxyurea + Concurrent chemoradiation: carboplatin followed byRadiation ABX: 100 mg/m² concomitant 5-FU: 600 mg/m² fluorouracil,hydroxyurea: 5000 mg BID hydroxyurea, Abraxane ® and IMRT for locallyadvanced head and neck cancers 9 ABX + ABX: 20-50 mg/m² qwk × 7 LocallyPhase I trial of Carboplatin + dose escalation Advanced Abraxane ® inErbitux ® + Eribitux ®: 400 mg/m² day 7, Head and combination withRadiation 250 mg/m² qwk × 7 Neck Cancer carboplatin, cetuximab Carbo:AUC = 1.5 qwk × 7 and IMRT in locally advanced squamous cell IMRT cancerof the head and neck 10 ABX + qwk Pancreatic A randomized phase IIGemcitabine + Cancer trial of weekly Radiation gemcitabine, Abraxane ®,and external irradiation for locally advanced pancreatic cancer 11 ABX +Gastric Phase I/II combination Cisplatin + Malignancies ofAbraxane ®/cisplatin Radiation and radiation for patients with resectedgastric/GEJ malignancies.

In some variations, the invention provides pharmaceutical compositionscomprising nanoparticles comprising a taxane (such as paclitaxel) and acarrier protein (such as albumin) for use in the treatment of aproliferative disease (such as cancer), wherein said use comprises asecond therapy comprising radiation therapy, surgery, or combinationsthereof.

Metronomic Therapy

The invention also provides metronomic therapy regime. There is provideda method of administering to an individual a composition comprisingnanoparticles comprising a taxane (such as paclitaxel, docetaxel, orortataxel) and a carrier protein (such as albumin) based on a metronomicdosing regime. The methods are applicable to methods of treatment,delaying development, and other clinical settings and configurationsdescribed herein. For example, in some variations, the methods areuseful for treatment of proliferative diseases (such as cancer).

“Metronomic dosing regime” used herein refers to frequent administrationof a taxane at without prolonged breaks at a dose below the establishedmaximum tolerated dose via a traditional schedule with breaks(hereinafter also referred to as a “standard MTD schedule” or a“standard MTD regime”). In metronomic dosing, the same, lower, or highercumulative dose over a certain time period as would be administered viaa standard MTD schedule may ultimately be administered. In some cases,this is achieved by extending the time frame and/or frequency duringwhich the dosing regime is conducted while decreasing the amountadministered at each dose. Generally, the taxane administered via themetronomic dosing regime of the present invention is better tolerated bythe individual. Metronomic dosing can also be referred to as maintenancedosing or chronic dosing.

In some variations, there is provided a method of administering acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), wherein the nanoparticle composition isadministered over a period of at least one month, wherein the intervalbetween each administration is no more than about a week, and whereinthe dose of the taxane at each administration is about 0.25% to about25% of its maximum tolerated dose following a traditional dosing regime.In some variations, there is provided a method of administering acomposition comprising nanoparticles comprising paclitaxel and analbumin, wherein the nanoparticle composition is administered over aperiod of at least one month, wherein the interval between eachadministration is no more than about a week, and wherein the dose of thetaxane at each administration is about 0.25% to about 25% of its maximumtolerated dose following a traditional dosing regime.

In some variations, the dosing of the taxane (such as paclitaxel) in thenanoparticle composition per administration is less than about any of1%, 2%, 3&, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 18%,20%, 22%, 24%, or 25% of the MTD for the same taxane (such aspaclitaxel) in the same formulation following a given traditional dosingschedule. Traditional dosing schedule refers to the dosing schedule thatis generally established in a clinical setting. For example, thetradition dosing schedule for Abraxane® is a three-weekly schedule,i.e., administering the composition every three weeks.

In some variations, the dosing of the taxane (such as paclitaxel) peradministration is between about 0.25% to about 25% of the correspondingMTD value, including for example any of about 0.25% to about 20%, about0.25% to about 15%, about 0.25% to about 10%, about 0.25% to about 20%,and about 0.25% to about 25%, of the corresponding MTD value. The MTDvalue for a taxane following a traditional dosing schedule is known orcan be easily determined by a person skilled in the art. For example,the MTD value when Abraxane® is administered following a traditionalthree-week dosing schedule is about 300 mg/m².

In some variations, there is provided a method of administering acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), wherein the nanoparticle composition isadministered over a period of at least one month, wherein the intervalbetween each administration is no more than about a week, and whereinthe dose of the taxane at each administration is about 0.25 mg/m² toabout 25 mg/m². In some variations, there is provided a method ofadministering a composition comprising nanoparticles comprisingpaclitaxel and an albumin, wherein the nanoparticle composition isadministered over a period of at least one month, wherein the intervalbetween each administration is no more than about a week, and whereinthe dose of the taxane at each administration is about 0.25 mg/m² toabout 25 mg/m².

In some variations, the dose of the taxane (such as paclitaxel) at eachadministration is less than about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 18, 20, 22, 25, and 30 mg/m². For example, the dose ofthe taxane (such as paclitaxel) can range from about 0.25 mg/m² to about30 mg/m², about 0.25 mg/m² to about 25 mg/m², about 0.25 mg/m² to about15 mg/m², about 0.25 mg/m² to about 10 mg/m², and about 0.25 mg/m² toabout 5 mg/m².

Dosing frequency for the taxane (such as paclitaxel) in the nanoparticlecomposition includes, but is not limited to, at least about any of oncea week, twice a week, three times a week, four times a week, five timesa week, six times a week, or daily. Typically, the interval between eachadministration is less than about a week, such as less than about any of6, 5, 4, 3, 2, or 1 day. In some variations, the interval between eachadministration is constant. For example, the administration can becarried out daily, every two days, every three days, every four days,every five days, or weekly. In some variations, the administration canbe carried out twice daily, three times daily, or more frequent.

The metronomic dosing regimes described herein can be extended over anextended period of time, such as from about a month up to about threeyears. For example, the dosing regime can be extended over a period ofany of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, and 36months. Generally, there are no breaks in the dosing schedule.

The cumulative dose of the taxane (such as paclitaxel) administered bythe metronomic regime may be higher than that of the taxane administeredaccording to a standard MTD dosing schedule over the same time period.In some variations, the cumulative dose of the taxane administered bythe metronomic regime equals to or is lower than that of the taxaneadministered according to a standard MTD dosing schedule over the sametime period.

It is understood that the teaching provided herein is for examples only,and that metronomic dosing regime can be routinely designed inaccordance with the teachings provided herein and based upon theindividual standard MTD schedule, and that the metronomic dosing regimeused in these experiments merely serves as one example of possiblechanges in dosing interval and duration which are made to a standard MTDschedule to arrive at an optimal metronomic dosing regime.

The metronomic dosing regime described herein may be used alone as atreatment of a proliferative disease, or carried out in a combinationtherapy context, such as the combination therapies described herein. Insome variations, the metronomic therapy dosing regime may be used incombination or conjunction with other established therapies administeredvia standard MTD regimes. By “combination or in conjunction with” it ismeant that the metronomic dosing regime of the present invention isconducted either at the same time as the standard MTD regime ofestablished therapies, or between courses of induction therapy tosustain the benefit accrued to the individual by the induction therapy,the intent is to continue to inhibit tumor growth while not undulycompromising the individual's health or the individual's ability towithstand the next course of induction therapy. For example, ametronomic dosing regime may be adopted after an initial short course ofMTD chemotherapy.

The nanoparticle compositions administered based on the metronomicdosing regime described herein can be administered to an individual(such as human) via various routes, such as parenterally, includingintravenous, intra-arterial, intrapulmonary, oral, inhalation,intravesicular, intramuscular, intra-tracheal, subcutaneous,intraocular, intrathecal, or transdermal. For example, the nanoparticlecomposition can be administered by inhalation to treat conditions of therespiratory tract. The composition can be used to treat respiratoryconditions such as pulmonary fibrosis, broncheolitis obliterans, lungcancer, bronchoalveolar carcinoma, and the like. In some variations, thenanoparticle composition is administered orally.

Some various exemplary variations are provided below.

In some variations, there is provided a method of administering acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), wherein the nanoparticle composition isadministered over a period of at least one month, wherein the intervalbetween each administration is no more than about a week, and whereinthe dose of the taxane at each administration is about 0.25% to about25% of its maximum tolerated dose following a traditional dosing regime.In some variations, the taxane is coated with the carrier protein (suchas albumin). In some variations, the dose of the taxane peradministration is less than about any of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 18%, 20%, 22%, 24%, or 25% of themaximum tolerated dose. In some variations, the taxane is administeredat least about any of 1×, 2×, 3×, 4×, 5×, 6×, 7× (i.e., daily) a week.In some variations, the intervals between each administration are lessthan about any of 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, and 1day. In some variations, the taxane is administered over a period of atleast about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30 and 36months.

In some variations, there is provided a method of administering acomposition comprising nanoparticles comprising paclitaxel and analbumin, wherein the nanoparticle composition is administered over aperiod of at least one month, wherein the interval between eachadministration is no more than about a week, and wherein the dose of thetaxane at each administration is about 0.25% to about 25% of its maximumtolerated dose following a traditional dosing regime. In somevariations, the paclitaxel/albumin nanoparticles have an averagediameter of no greater than about 200 nm. In some variations, thepaclitaxel/albumin nanoparticle composition is substantially free (suchas free) of surfactant (such as Cremophor). In some variations, theweight ratio of the albumin to paclitaxel in the composition is about18:1 or less, such as about 9:1 or less. In some variations, thepaclitaxel is coated with albumin. In some variations, thepaclitaxel/albumin nanoparticles have an average diameter of no greaterthan about 200 nm and the paclitaxel/albumin composition issubstantially free (such as free) of surfactant (such as Cremophor). Insome variations, the paclitaxel/albumin nanoparticles have an averagediameter of no greater than about 200 nm and the paclitaxel is coatedwith albumin. In some variations, the nanoparticle composition isAbraxane®

In some variations, there is provided a method of administering acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), wherein the nanoparticle composition isadministered over a period of at least one month, wherein the intervalbetween each administration is no more than about a week, and whereinthe dose of the taxane at each administration is about 0.25 mg/m² toabout 25 mg/m². In some variations, the taxane is coated with thecarrier protein (such as albumin). In some variations, the dose of thetaxane per administration is less than about any of 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 18, 20, 22, and 25 mg/m². In some variations,the taxane is administered at least about any of 1×, 2×, 3×, 4×, 5×, 6×,7× (i.e., daily) a week. In some variations, the intervals between eachadministration are less than about any of 7 days, 6 days, 5 days, 4days, 3 days, 2 days, and 1 day. In some variations, the taxane isadministered over a period of at least about any of 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 18, 24, 30 and 36 months.

In some variations, there is provided a method of administering acomposition comprising nanoparticles comprising paclitaxel and analbumin, wherein the nanoparticle composition is administered over aperiod of at least one month, wherein the interval between eachadministration is no more than about a week, and wherein the dose of thetaxane at each administration is about 0.25 mg/m² to about 25 mg/m². Insome variations, the paclitaxel/albumin nanoparticles have an averagediameter of no greater than about 200 nm. In some variations, thepaclitaxel/albumin nanoparticle composition is substantially free (suchas free) of surfactant (such as Cremophor). In some variations, theweight ratio of the albumin to paclitaxel in the composition is about18:1 or less, such as about 9:1 or less. In some variations, thepaclitaxel is coated with albumin. In some variations, thepaclitaxel/albumin nanoparticles have an average diameter of no greaterthan about 200 nm and the paclitaxel/albumin composition issubstantially free (such as free) of surfactant (such as Cremophor). Insome variations, the paclitaxel/albumin nanoparticles have an averagediameter of no greater than about 200 nm and the paclitaxel is coatedwith albumin. In some variations, the nanoparticle composition isAbraxane®

In some variations, the Abraxane® (or other paclitaxel/albuminnanoparticle compositions) is administered at the dose of about 3 mg/kgto about 10 mg/kg daily. In some variations, the Abraxane® isadministered at the dose of about 6 mg/kg to about 10 mg/kg daily. Insome variations, the Abraxane® is administered at the dose of about 6mg/kg daily. In some variations, Abraxane® is administered at the doseof about 3 mg/kg daily.

The invention also provides compositions for use in the metronomicregime(s) described herein. In some variations, there is provided acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), wherein said composition is administered toan individual via a metronomic dosing regime, such as the dosing regimedescribed herein.

Other Aspects of the Invention

In another aspects, there are provided methods of treating proliferativediseases comprising administering a composition comprising nanoparticlescomprising a taxane (including paclitaxel, docetaxel, or ortataxel) anda carrier protein (such as albumin). In some variations, there isprovided a method of treating cancer comprising administering acomposition comprising nanoparticles comprising ortataxel and a carrierprotein (such as albumin).

In some variations, there is provided methods of treating proliferativediseases comprising administering a composition comprising nanoparticlescomprising a thiocolchicine or its derivative (such as dimericthiocolchicine) and a carrier protein (such as albumin). In somevariations, there is provided a method of treating cancer comprisingadministering a composition comprising nanoparticles comprising dimericcolchicines and a carrier protein (such as albumin). In some variations,the nanoparticle composition is any of (and in some variations selectedfrom the group consisting of) Nab-5404, Nab-5800, and Nab-5801.

In some variations, there is provided a method of treating cancercomprising administering a composition comprising nanoparticlescomprising paclitaxel, wherein the nanoparticle composition isadministered according to any of the dosing regimes described in Table3. In some variations, the cancer is a Taxane refractory metastaticbreast cancer.

TABLE 3 Row Study therapy No. Combination Regimen/Dosage type Protocoltitle 1. ABX alone ABX: 125 mg/m² qwk × 3/4 Metastatic Phase II studywith Breast Cancer weekly Abraxane ® treatment in taxane- refractory MBCpatients 2. ABX alone Arm 1: ABX 130 mg/m² qwk Metastatic 3-arm phase IItrial in 1st- Arm 2: ABX 260 mg/m² q2wk Breast Cancer line Her-2- MBCpatients. Arm 3: ABX 260 mg/m² q3wk 3. ABX alone ABX: 260 mg/m² q3wkMetastatic Phase II Controlled, (Capxol) vs Breast Cancer Randomized,Open Label Taxol: 175 mg/m² q3wk Study to Evaluate the Efficacy andSafety of Capxol (a Cremophor- Free Nanoparticle Paclitaxel) andcremophor-formulated paclitaxel injection in Patient with MetastaticBreast Cancer 4. ABX alone Arm 1: ABX weekly Metastatic 3-arm phase IItrial in 1st- Arm 2: ABX q3wk Breast Cancer line and 2nd-line MBC, Arm3: Taxol weekly with biological correlates analysis 5. ABX alone ABX:300 mg/m² q3wk Stage IIA, IIB, Phase II trial of IIIA, IIIB andneoadjuvant IV breast cancer chemotherapy (NCT) with nanoparticlepaclitaxel (ABI-007, Abraxane ®) in women with clinical stage IIA, IIB,IIIA, IIIB and IV (with intact primary) breast cancers 6. ABX alone ABX:125 mg/m² qwk × 3/4 1st-line Phase I/II study of advanced Abraxane ®monotherapy NSCLC in 1st-line advanced NSCLC 7. ABX alone ABX 260 mg/m²1st-line NSCLC Phase II ABX mono in q3wk 1st-line NSCLC 8. ABX alone Arm1: ABX q3wk 2^(nd) line NSCLC Phase II study of Arm 2: ABX qwkAbraxane ® monotherapy Doses TBD in 2^(nd)-line NSCLC 9. ABX alone ABX:100 mg/m² qwk Prostate Cancer Randomized phase II vs study Abraxane ®weekly ABX: 260 mg/m² q3wk vs every three weeks in front line HRP 10.ABX alone ABX qwk Prostate Cancer Phase II Abraxane ® in 1st- lineprostate cancer 11. ABX alone ABX: 150 mg/m² qwk × 3/4 for 2 ProstateCancer Phase II neoadjuvant cycles study 12. ABX alone ABX: 100 mg/m²qwk (no break) Prostate Cancer Phase II Abraxane ® 100 mg weekly nobreak 13. ABX alone ABX: 100 mg/m² (previously Malignant Phase IIpreviously treated treated) Melanoma and untreated metastatic ABX: 150mg/m² (untreated) melanoma patients qwk × 3/4 14. ABX alone ABX: 125mg/m² Carcinoma of Phase II study of qwk × 3/4 the cervix Abraxane ® intreatment of persistent or recurrent carcinoma of the cervix 15. ABXalone Ovarian Cancer Phase II study of Abraxane ® for treatment ofadvanced ovarian cancer (3^(rd) line) 16. ABX alone non-hematologicPhase II single treatment (ABI-007) malignancies use of ABI-007(Abraxane ®) for the treatment of non- hematologic malignancies.Compassionate use

Nanoparticle Compositions

The nanoparticle compositions described herein comprise nanoparticlescomprising (in various variations consisting essentially of) a taxane(such as paclitaxel) and a carrier protein (such as albumin).Nanoparticles of poorly water soluble drugs (such as taxane) have beendisclosed in, for example, U.S. Pat. Nos. 5,916,596; 6,506,405; and6,537,579 and also in U.S. Pat. Pub. No. 2005/0004002A1. Although thedescription provided below is specific to taxane, it is understood thatthe same applies to other drugs, such as rapamycin, 17-AAG, and dimericthiocolchicine.

In some variations, the composition comprises nanoparticles with anaverage or mean diameter of no greater than about 1000 nanometers (nm),such as no greater than about any of 900, 800, 700, 600, 500, 400, 300,200, and 100 nm. In some variations, the average or mean diameters ofthe nanoparticles is no greater than about 200 nm. In some variations,the average or mean diameters of the nanoparticles is no greater thanabout 150 nm. In some variations, the average or mean diameters of thenanoparticles is no greater than about 100 nm. In some variations, theaverage or mean diameter of the nanoparticles is about 20 to about 400nm. In some variations, the average or mean diameter of thenanoparticles is about 40 to about 200 nm. In some variations, thenanoparticles are sterile-filterable.

The nanoparticles described herein may be present in a dry formulation(such as lyophilized composition) or suspended in a biocompatiblemedium. Suitable biocompatible media include, but are not limited to,water, buffered aqueous media, saline, buffered saline, optionallybuffered solutions of amino acids, optionally buffered solutions ofproteins, optionally buffered solutions of sugars, optionally bufferedsolutions of vitamins, optionally buffered solutions of syntheticpolymers, lipid-containing emulsions, and the like.

The term “proteins” refers to polypeptides or polymers of amino acids ofany length (including full length or fragments), which may be linear orbranched, comprise modified amino acids, and/or be interrupted bynon-amino acids. The term also encompasses an amino acid polymer thathas been modified naturally or by intervention; for example, disulfidebond formation, glycosylation, lipidation, acetylation, phosphorylation,or any other manipulation or modification. Also included within thisterm are, for example, polypeptides containing one or more analogs of anamino acid (including, for example, unnatural amino acids, etc.), aswell as other modifications known in the art. The proteins describedherein may be naturally occurring, i.e., obtained or derived from anatural source (such as blood), or synthesized (such as chemicallysynthesized or by synthesized by recombinant DNA techniques).

Examples of suitable carrier proteins include proteins normally found inblood or plasma, which include, but are not limited to, albumin,immunoglobulin including IgA, lipoproteins, apolipoprotein B, alpha-acidglycoprotein, beta-2-macroglobulin, thyroglobulin, transferin,fibronectin, factor VII, factor VIII, factor IX, factor X, and the like.In some variations, the carrier protein is non-blood protein, such ascasein, α-lactalbumin, and P-lactoglobulin. The carrier proteins mayeither be natural in origin or synthetically prepared. In somevariations, the pharmaceutically acceptable carrier comprises albumin,such as human serum albumin. Human serum albumin (HSA) is a highlysoluble globular protein of M, 65K and consists of 585 amino acids. HSAis the most abundant protein in the plasma and accounts for 70-80% ofthe colloid osmotic pressure of human plasma. The amino acid sequence ofHSA contains a total of 17 disulphide bridges, one free thiol (Cys 34),and a single tryptophan (Trp 214). Intravenous use of HSA solution hasbeen indicated for the prevention and treatment of hypovolumic shock(see, e.g., Tullis, JAMA, 237, 355-360, 460-463, (1977)) and Houser etal., Surgery, Gynecology and Obstetrics, 150, 811-816 (1980)) and inconjunction with exchange transfusion in the treatment of neonatalhyperbilirubinemia (see, e.g., Finlayson, Seminars in Thrombosis andHemostasis, 6, 85-120, (1980)). Other albumins are contemplated, such asbovine serum albumin. Use of such non-human albumins could beappropriate, for example, in the context of use of these compositions innon-human mammals, such as the veterinary (including domestic pets andagricultural context).

Human serum albumin (HSA) has multiple hydrophobic binding sites (atotal of eight for fatty acids, an endogenous ligand of HSA) and binds adiverse set of taxanes, especially neutral and negatively chargedhydrophobic compounds (Goodman et al., The Pharmacological Basis ofTherapeutics, 9^(th) ed, McGraw-Hill New York (1996)). Two high affinitybinding sites have been proposed in subdomains IIA and IIIA of HSA,which are highly elongated hydrophobic pockets with charged lysine andarginine residues near the surface which function as attachment pointsfor polar ligand features (see, e.g., Fehske et al., Biochem. Pharmcol.,30, 687-92 (198a), Vorum, Dan. Med. Bull., 46, 379-99 (1999),Kragh-Hansen, Dan. Med. Bull., 1441, 131-40 (1990), Curry et al., Nat.Struct. Biol., 5, 827-35 (1998), Sugio et al., Protein. Eng., 12, 439-46(1999), He et al., Nature, 358, 209-15 (199b), and Carter et al., Adv.Protein. Chem., 45, 153-203 (1994)). Paclitaxel and propofol have beenshown to bind HSA (see, e.g., Paal et al., Eur. J. Biochem., 268(7),2187-91 (200a), Purcell et al., Biochim. Biophys. Acta, 1478(a), 61-8(2000), Altmayer et al., Arzneimittelforschung, 45, 1053-6 (1995), andGarrido et al., Rev. Esp. Anestestiol. Reanim., 41, 308-12 (1994)). Inaddition, docetaxel has been shown to bind to human plasma proteins(see, e.g., Urien et al., Invest. New Drugs, 14(b), 147-51 (1996)).

The carrier protein (such as albumin) in the composition generallyserves as a carrier for the taxane, i.e., the carrier protein in thecomposition makes the taxane more readily suspendable in an aqueousmedium or helps maintain the suspension as compared to compositions notcomprising a carrier protein. This can avoid the use of toxic solvents(or surfactants) for solubilizing the taxane, and thereby can reduce oneor more side effects of administration of the taxane into an individual(such as a human). Thus, in some variations, the composition describedherein is substantially free (such as free) of surfactants, such asCremophor (including Cremophor EL® (BASF)). In some variations, thenanoparticle composition is substantially free (such as free) ofsurfactants. A composition is “substantially free of Cremophor” or“substantially free of surfactant” if the amount of Cremophor orsurfactant in the composition is not sufficient to cause one or moreside effect(s) in an individual when the nanoparticle composition isadministered to the individual.

The amount of carrier protein in the composition described herein willvary depending on other components in the composition. In somevariations, the composition comprises a carrier protein in an amountthat is sufficient to stabilize the taxane in an aqueous suspension, forexample, in the form of a stable colloidal suspension (such as a stablesuspension of nanoparticles). In some variations, the carrier protein isin an amount that reduces the sedimentation rate of the taxane in anaqueous medium. For particle-containing compositions, the amount of thecarrier protein also depends on the size and density of nanoparticles ofthe taxane.

A taxane is “stabilized” in an aqueous suspension if it remainssuspended in an aqueous medium (such as without visible precipitation orsedimentation) for an extended period of time, such as for at leastabout any of 0.1, 0.2, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,24, 36, 48, 60, or 72 hours. The suspension is generally, but notnecessarily, suitable for administration to an individual (such ashuman). Stability of the suspension is generally (but not necessarily)evaluated at a storage temperature (such as room temperature (such as20-25° C.) or refrigerated conditions (such as 4° C.)). For example, asuspension is stable at a storage temperature if it exhibits noflocculation or particle agglomeration visible to the naked eye or whenviewed under the optical microscope at 1000 times, at about fifteenminutes after preparation of the suspension. Stability can also beevaluated under accelerated testing conditions, such as at a temperaturethat is higher than about 40° C.

In some variations, the carrier protein is present in an amount that issufficient to stabilize the taxane in an aqueous suspension at a certainconcentration. For example, the concentration of the taxane in thecomposition is about 0.1 to about 100 mg/ml, including for example anyof about 0.1 to about 50 mg/ml, about 0.1 to about 20 mg/ml, about 1 toabout 10 mg/ml, about 2 mg/ml to about 8 mg/ml, about 4 to about 6mg/ml, about 5 mg /ml. In some variations, the concentration of thetaxane is at least about any of 1.3 mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml,4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 15mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, and 50 mg/ml. In somevariations, the carrier protein is present in an amount that avoids useof surfactants (such as Cremophor), so that the composition is free orsubstantially free of surfactant (such as Cremophor).

In some variations, the composition, in liquid form, comprises fromabout 0.1% to about 50% (w/v) (e.g. about 0.5% (w/v), about 5% (w/v),about 10% (w/v), about 15% (w/v), about 20% (w/v), about 30% (w/v),about 40% (w/v), or about 50% (w/v)) of carrier protein. In somevariations, the composition, in liquid form, comprises about 0.5% toabout 5% (w/v) of carrier protein.

In some variations, the weight ratio of carrier protein, e.g., albumin,to the taxane in the nanoparticle composition is such that a sufficientamount of taxane binds to, or is transported by, the cell. While theweight ratio of carrier protein to taxane will have to be optimized fordifferent carrier protein and taxane combinations, generally the weightratio of carrier protein, e.g., albumin, to taxane (w/w) is about 0.01:1to about 100:1, about 0.02:1 to about 50:1, about 0.05:1 to about 20:1,about 0.1:1 to about 20:1, about 1:1 to about 18:1, about 2:1 to about15:1, about 3:1 to about 12:1, about 4:1 to about 10:1, about 5:1 toabout 9:1, or about 9:1. In some variations, the carrier protein totaxane weight ratio is about any of 18:1 or less, 15:1 or less, 14:1 orless, 13:1 or less, 12:1 or less, 11:1 or less, 10:1 or less, 9:1 orless, 8:1 or less, 7:1 or less, 6:1 or less, 5:1 or less, 4:1 or less,and 3:1 or less.

In some variations, the carrier protein allows the composition to beadministered to an individual (such as human) without significant sideeffects. In some variations, the carrier protein (such as albumin) is inan amount that is effective to reduce one or more side effects ofadministration of the taxane to a human. The term “reducing one or moreside effects of administration of the taxane” refers to reduction,alleviation, elimination, or avoidance of one or more undesirableeffects caused by the taxane, as well as side effects caused by deliveryvehicles (such as solvents that render the taxanes suitable forinjection) used to deliver the taxane. Such side effects include, forexample, myelosuppression, neurotoxicity, hypersensitivity,inflammation, venous irritation, phlebitis, pain, skin irritation,peripheral neuropathy, neutropenic fever, anaphylactic reaction, venousthrombosis, extravasation, and combinations thereof. These side effects,however, are merely exemplary and other side effects, or combination ofside effects, associated with taxanes can be reduced.

In some variations, the composition comprises Abraxane®. Abraxane® is aformulation of paclitaxel stabilized by human albumin USP, which can bedispersed in directly injectable physiological solution. When dispersedin a suitable aqueous medium such as 0.9% sodium chloride injection or5% dextrose injection, Abraxane® forms a stable colloidal suspension ofpaclitaxel. The mean particle size of the nanoparticles in the colloidalsuspension is about 130 nanometers. Since HSA is freely soluble inwater, Abraxane® can be reconstituted in a wide range of concentrationsranging from dilute (0.1 mg/ml paclitaxel) to concentrated (20 mg/mlpaclitaxel), including for example about 2 mg/ml to about 8 mg/ml, about5 mg/ml.

Methods of making nanoparticle compositions are known in the art. Forexample, nanoparticles containing taxanes (such as paclitaxel) andcarrier protein (such as albumin) can be prepared under conditions ofhigh shear forces (e.g., sonication, high pressure homogenization, orthe like). These methods are disclosed in, for example, U.S. Pat. Nos.5,916,596; 6,506,405; and 6,537,579 and also in U.S. Pat. Pub. No.2005/0004002A1.

Briefly, the taxane (such as docetaxel) is dissolved in an organicsolvent, and the solution can be added to a human serum albuminsolution. The mixture is subjected to high pressure homogenization. Theorganic solvent can then be removed by evaporation. The dispersionobtained can be further lyophilized. Suitable organic solvent include,for example, ketones, esters, ethers, chlorinated solvents, and othersolvents known in the art. For example, the organic solvent can bemethylene chloride and chloroform/ethanol (for example with a ratio of1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, or 9:a).

Other Components in the Nanoparticle Compositions

The nanoparticles described herein can be present in a composition thatinclude other agents, excipients, or stabilizers. For example, toincrease stability by increasing the negative zeta potential ofnanoparticles, certain negatively charged components may be added. Suchnegatively charged components include, but are not limited to bile saltsof bile acids consisting of glycocholic acid, cholic acid,chenodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid,taurochenodeoxycholic acid, litocholic acid, ursodeoxycholic acid,dehydrocholic acid and others; phospholipids including lecithin (eggyolk) based phospholipids which include the followingphosphatidylcholines: palmitoyloleoylphosphatidylcholine,palmitoyllinoleoylphosphatidylcholine,stearoyllinoleoylphosphatidylcholine stearoyloleoylphosphatidylcholine,stearoylarachidoylphosphatidylcholine, anddipalmitoylphosphatidylcholine. Other phospholipids includingL-α-dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine(DOPC), distearyolphosphatidylcholine (DSPC), hydrogenated soyphosphatidylcholine (HSPC), and other related compounds. Negativelycharged surfactants or emulsifiers are also suitable as additives, e.g.,sodium cholesteryl sulfate and the like.

In some variations, the composition is suitable for administration to ahuman. In some variations, the composition is suitable foradministration to a mammal such as, in the veterinary context, domesticpets and agricultural animals. There are a wide variety of suitableformulations of the nanoparticle composition (see, e.g., U.S. Pat. Nos.5,916,596 and 6,096,331). The following formulations and methods aremerely exemplary and are in no way limiting. Formulations suitable fororal administration can consist of (a) liquid solutions, such as aneffective amount of the compound dissolved in diluents, such as water,saline, or orange juice, (b) capsules, sachets or tablets, eachcontaining a predetermined amount of the active ingredient, as solids orgranules, (c) suspensions in an appropriate liquid, and (d) suitableemulsions. Tablet forms can include one or more of lactose, mannitol,corn starch, potato starch, microcrystalline cellulose, acacia, gelatin,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, stearic acid, and other excipients, colorants, diluents,buffering agents, moistening agents, preservatives, flavoring agents,and pharmacologically compatible excipients. Lozenge forms can comprisethe active ingredient in a flavor, usually sucrose and acacia ortragacanth, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to the activeingredient, such excipients as are known in the art.

Examples of suitable carriers, excipients, and diluents include, but arenot limited to, lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,cellulose, water, saline solution, syrup, methylcellulose, methyl- andpropylhydroxybenzoates, talc, magnesium stearate, and mineral oil. Theformulations can additionally include lubricating agents, wettingagents, emulsifying and suspending agents, preserving agents, sweeteningagents or flavoring agents.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation compatible with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described. Injectable formulations are preferred.

In some variations, the composition is formulated to have a pH range ofabout 4.5 to about 9.0, including for example pH ranges of any of about5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0. Insome variations, the pH of the composition is formulated to no less thanabout 6, including for example no less than about any of 6.5, 7, or 8(such as about 8). The composition can also be made to be isotonic withblood by the addition of a suitable tonicity modifier, such as glycerol.

Kits

The invention also provides kits for use in the instant methods. Kits ofthe invention include one or more containers comprisingtaxane-containing nanoparticle compositions (or unit dosage forms and/orarticles of manufacture) and/or a chemotherapeutic agent, and in somevariations, further comprise instructions for use in accordance with anyof the methods described herein. The kit may further comprise adescription of selection an individual suitable or treatment.Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable.

In some variations, the kit comprises a) a composition comprisingnanoparticles comprising a taxane and a carrier protein (such asalbumin), b) an effective amount of at least one other chemotherapeuticagent, and c) instructions for administering the nanoparticles and thechemotherapeutic agents simultaneously and/or sequentially, fortreatment of a proliferative disease (such as cancer). In somevariations, the taxane is any of paclitaxel, docetaxel, and ortataxel.In some variations, the kit comprises nanoparticles comprising a) acomposition comprising nanoparticles comprising paclitaxel and analbumin (such as Abraxane®), b) an effective amount of at least oneother chemotherapeutic agent, and c) instructions for administering thenanoparticles and the chemotherapeutic agents simultaneously and/orsequentially, for the effective treatment of a proliferative disease(such as cancer).

In some variations, the kit comprises a) a composition comprisingnanoparticles comprising a taxane and a carrier protein (such asalbumin), b) a composition comprising nanoparticles comprising at leastone other chemotherapeutic agent and a carrier protein (such asalbumin), and c) instructions for administering the nanoparticlecompositions simultaneously and/or sequentially, for treatment of aproliferative disease (such as cancer). In some variations, the kitcomprises nanoparticles comprising a) a composition comprisingnanoparticles comprising paclitaxel and an albumin (such as Abraxane®),b) a composition comprising nanoparticles comprising at least one otherchemotherapeutic agent and a carrier protein (such as albumin), and c)instructions for administering the nanoparticle compositionssimultaneously and/or sequentially, for the effective treatment of aproliferative disease (such as cancer).

The nanoparticles and the chemotherapeutic agents can be present inseparate containers or in a single container. It is understood that thekit may comprise one distinct composition or two or more compositionswherein one composition comprises nanoparticles and one compositioncomprises a chemotherapeutic agent.

The kits of the invention are in suitable packaging. Suitable packaginginclude, but is not limited to, vials, bottles, jars, flexible packaging(e.g., seled Mylar or plastic bags), and the like. Kits may optionallyprovide additional components such as buffers and interpretativeinformation.

The instructions relating to the use of the nanoparticle compositionsgenerally include information as to dosage, dosing schedule, and routeof administration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Forexample, kits may be provided that contain sufficient dosages of thetaxane (such as taxane) as disclosed herein to provide effectivetreatment of an individual for an extended period, such as any of aweek, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5months, 7 months, 8 months, 9 months, or more. Kits may also includemultiple unit doses of the taxane and pharmaceutical compositions andinstructions for use and packaged in quantities sufficient for storageand use in pharmacies, for example, hospital pharmacies and compoundingpharmacies.

Those skilled in the art will recognize that several variations arepossible within the scope and spirit of this invention. The inventionwill now be described in greater detail by reference to the followingnon-limiting examples. The following examples further illustrate theinvention but, of course, should not be construed as in any way limitingits scope.

EXAMPLES Example 1 Improved Response and Reduced Toxicities forAbraxane® Compared to Taxol® in a Phase III Study of Abraxane® GivenEvery Three Weeks

Significantly reduced incidence of neutropenia and hypersensitivity,absence of requirement of steroid premedication, shorter duration ofneuropathy, shorter infusion time and higher dose.

ABI-007 (Abraxane®), the first biologically interactive albumin-boundpaclitaxel in a nanoparticle form, free of any solvent, was comparedwith Cremophor -based paclitaxel (Taxol™) in individuals with metastaticbreast cancer (MBC). This phase III study was performed to confirm thepreclinical studies demonstrating superior efficacy and reduced toxicityof ABI-007 when compared with Taxol®. Individuals were randomly assignedto 3-week cycles of either ABI-007 260 mg/m² (iv) over 30 minuteswithout premedication (n=229) or Taxol® 175 mg/m² IV over 3 hours withpremedication (n=225). ABI-007 demonstrated significantly higherresponse rates compared with Taxol® (33% vs. 19%; p=0.001) andsignificantly longer time to tumor progression (23.0 vs. 16.9 weeks;HR=0.75; p=0.006). There was a trend for longer overall survival inindividuals who received ABI-007 (65.0 vs. 55.7 weeks; p 0.374). In anunplanned analysis, ABI-007 improved survival in individuals receivingtreatment as second- or greater-line therapy (56.4 vs. 46.7 weeks;HR=0.73; p=0.024). The incidence of grade 4 neutropenia wassignificantly lower in the ABI-007 group (9% vs. 22%; p<0.001) despite a49% higher paclitaxel dose. Grade 3 sensory neuropathy was more commonin the ABI-007 group than in the Taxol® group (10% vs. 2%; p<0.001) butwas easily managed and improved more rapidly (median, 22 days) than forTaxol® (median 73 days). No severe (grade 3 or 4) treatment-relatedhypersensitivity reactions occurred in any of the individuals in theABI-007 group despite the absence of premedication and shorteradministration time. In contrast, grade 3 hypersensitivity reactionsoccurred in the Taxol® group despite standard premedication (chest pain:2 individuals; allergic reaction: 3 individuals). Per protocol,corticosteroids and antihistamines were not administered routinely toindividuals in the ABI-007 group; however, premedication wasadministered for emesis, myalgia/arthralgia, or anorexia in 18individuals (8%) in the ABI-007 group in 2% of the treatment cycles,whereas 224 individuals (>99%) in the Taxol® group receivedpremedication at 95% of the cycles. The only clinical chemistry valuethat was notably different between the 2 treatment arms was higher serumglucose levels in the Taxol®-treated individuals, who also had a higherincidence of hyperglycemia reported as an AE (adverse effects) (15 [7%]vs. 3 [1%]; p=0.003). Overall, ABI-007 demonstrated greater efficacy anda favorable safety profile compared with Taxol® in this individualpopulation. The improved therapeutic index and elimination of thesteroid premedication required for solvent-based taxanes make thisnanoparticle albumin-bound paclitaxel an important advance in thetreatment of MBC.

Example 2 Weekly Abraxane® in Taxane-Refractory Metastatic Breast CancerIndividuals

A recent Phase II clinical study showed that weekly administration ofAbraxane® (nanoparticle albumin-bound paclitaxel) at a dose of 125 mg/m²resulted in long-term disease control in individuals with metastaticbreast cancer whose disease had progressed while being treated withTaxol® or Taxotere® (that is, individuals who are taxane-refractory).

Abraxane® is believed to represent the first biologically interactivecomposition that exploits the receptor-mediated (gp60) pathway found tobe integral to achieving high intracellular tumor concentrations of theactive ingredient—paclitaxel. The Phase II study included 75 individualswith taxane-refractory metastatic breast cancer. Abraxane® wasadministered weekly via a 30-minute infusion at 125 mg/m² withoutsteroid/antihistamine premedication or G-CSF prophylaxis. Individualsreceived three weekly doses followed by one week of rest, repeated every28 days. Unlike Taxol® or Taxotere®, which contain detergents that mayinhibit tumor uptake, the mechanism of action of the albumin-boundnanoparticle paclitaxel may result in improved outcomes, especially inthis difficult-to-treat individual population.

Specifically, the data showed that despite this high weekly dose of 125mg/m² in this highly pre-treated and prior taxane-exposed individualpopulation, only 3 of 75 individuals (4%) had to discontinue Abraxane®due to peripheral neuropathy. Furthermore, of those who experiencedGrade 3 peripheral neuropathy, 80% were typically able to resumetreatment after a delay of only 1 or 2 weeks and continued to receiveAbraxane® at a reduced dose for an average of 4 additional months. Thisrapid improvement was consistent with our observation from the Phase IIItrial—that the peripheral neuropathy induced by paclitaxel alone (i.e.,without Cremophor®) improves rapidly as compared to that induced byTaxol®. These Abraxane® clinical trial experiences provide the firstclinical opportunity to evaluate the effects of the chemotherapeuticagent itself, paclitaxel, from the effects from those of solvents. Basedupon both the Phase II and III experience, the data now suggest that theperipheral neuropathy from Abraxane® is not comparable to the peripheralneuropathy from Taxol® or Taxotere® with respect to duration and impacton the individual.

With regard to the clinical experience of peripheral neuropathyfollowing Taxol® or Taxotere®, Abraxis Oncology recently completed asurvey of 200 oncologists who were asked how long they thought theperipheral neuropathy induced by Taxol® took to improve and/or resolve:25% reported “7-12 months” and another 23% reported “never resolved”;for Taxotere®, the respective percentages were 29% and 7%. These dataare consistent with the statements in the Taxotere® and Taxol® packageinserts.

Analysis of the Phase II data demonstrates Abraxane® to be active inthis poor-prognosis individual population (87% visceral (lung and liver)disease, 69%>3 metastatic sites, 88% tumor growth while on taxanes), oftaxane-refractory individuals with metastatic breast cancer.Observations included a 44% disease control in Taxotere®-refractoryindividuals and 39% disease control in Taxol®-refractory individuals. Ofthose individuals whose disease progressed while on Taxotere® alone inthe metastatic setting (n=27) a 19% response rate was noted afterreceiving weekly Abraxane®. Of those individuals whose diseaseprogressed while on Taxol® alone in the metastatic setting (n=23) a 13%response rate was noted after receiving weekly Abraxane®.

Abraxane® was found to be well tolerated when administered weekly over30 minutes without steroids or G-CSF prophylaxis: Grade 4 neutropenia=3%(without G-CSF); Grade 4 anemia=1%; no severe hypersensitivity reactions(despite absence of premedication). In this heavily pretreatedindividual population, 75% of individuals were treated at the full highdose of 125 mg/m² weekly Abraxane , with no dose reductions due totoxicities/adverse events. Of the individuals who developed grade 3sensory neuropathy, 77% were able to restart Abraxane at a reduced dose(75-100 mg/m²) and received a mean of 12.2 (range, 1-28) additionaldoses of Abraxane®. It was remarkable to note that of these individualswho resumed Abraxane®, 80% (8 of 10) were able to restart the drugwithin 14 days after improvement of neuropathy to Grade 1 or 2. Theseresults support the observations in the pivotal Phase III trial of 260mg/m² Abraxane® administered every 3 weeks, in which rapid improvementof neuropathy (median of 22 days) was also noted. Taken together thesetwo clinical trials suggest when paclitaxel is given alone, theneuropathy which occurs appears to be short-lived and is easily managed.

Abraxane® utilizes the gp60 receptor based pathway on the microvesselendothelial cells to transport the albumin-paclitaxel complex out of theblood vessel and into the tumor interstitium, and it has been shown thatTaxol® was not transported by this mechanism. Furthermore, analbumin-binding protein, SPARC, was over-expressed in breast tumors andmay play a role in the increased intra-tumoral accumulation ofAbraxane®. The proposed mechanism suggested that once in the tumorinterstitium, the albumin-paclitaxel complex would bind to SPARC thatwas present on the tumor cell surface and be rapidly internalized intothe tumor cell by a non-lysosomal mechanism.

In addition, the surfactants/solvents commonly used in current taxaneformulations such as Cremophor®, Tween® 80 and TPGS, strongly inhibitthe binding of paclitaxel to albumin, thereby limiting transendothelialtransport. Additional data presented showed a statistically improvedefficacy of Abraxane® over Taxotere® in the MX-1 mammary breastcarcinoma xenograft at equal dose.

In conclusion, 75% of individuals were treated at full high dose with nodose reductions. Data indicate rapid improvement of peripheralneuropathy when nanoparticle albumin-bound paclitaxel is administeredalone, without the solvent Cremophor®. Additional data provide increasedevidence that mechanism of action may play important role in enhancingindividual outcomes.

Example 3 Abraxane® (ABI-007) Acts Synergistically with TargetedAntiangiogenic Pro-Apoptotic Peptides (HKP) in MDA-MB-435 Human TumorXenografts

The antiangiogenic activity of small synthetic pro-apoptotic peptidescomposed of two functional domains, one targeting the CD13 receptors(aminopeptidase N) on tumor microvessels and the other disrupting themitochondrial membrane following internalization have previously beenreported. See Nat. Med. 1999 September; 5(9):1032-8. A second generationdimeric peptide, CNGRC-GG-d(KLAKLAK)₂, named HKP (Hunter Killer Peptide)was found to have improved antitumor activity. Since anti-angiogenicagents such as Avastin® exhibit synergism in combination with cytotoxicagents such as 5-fluorouracil, we evaluated the combination of theantiangiogenic HKP with Abraxane® (ABI-007), an albumin nanoparticlepaclitaxel that is transported by the gp60 receptor in vascularendothelium (Desai, SABCS 2003), in MDA-MB-435 human breast tumorxenografts.

Methods: MDA-MB-435 human tumor xenografts were established at anaverage tumor volume of 100 mm³, mice were randomized into groups of12-13 animals and treated with HKP, Abraxane®, or HKP and Abraxane®. HKPwas delivered i.v. (250 ug), once a week, for 16 weeks. Abraxane® wasadministered i.v., daily for 5 days at 10 mg/kg/day only for the firstweek of treatment. The Abraxane® dose used was substantially below itsMTD (30 mg/kg/day, qd×5) to prevent the tumor from complete regressionso effect of HKP could be noted.

Results: At nineteen weeks of treatment, tumor volume was significantlydecreased between control group (10,298 mm³+2,570) and HKP (4,372mm³+2,470; p <0.05 vs control) or ABI-007 (3,909 mm³+506; p<0.01 vscontrol). The combination of ABI-007 and HKP significantly reduced thetumor volume over either monotherapy (411 mm³±386; p<0.01 vs. Abraxane®monotherapy or HKP monotherapy). The treatments were well tolerated.

Conclusion: The combination of Abraxane® (ABI-007), a nanoparticlealbumin-bound paclitaxel, with the vascular targeting anti-angiogenicdimeric peptide HKP (CNGRC-GG-d(KLAKLAK)₂) against the MDA-MB-435xenograft breast tumor showed a significant reduction in tumor volumecompared to monotherapy of either agent alone. Our results suggest thatthe combination of Abraxane® with antiangiogenic agents such as HKPs orperhaps Avastin® may be beneficial.

Example 4 Metronomic ABI-007 Therapy: Antiangiogenic and AntitumorActivity of a Nanoparticle Albumin-Bound Paclitaxel Example 4a

Methods: The antiangiogenic activity of ABI-007 was assessed by the rataortic ring, human umbilical vein endothelial cell (HUVEC) proliferationand tube formation assays. Optimal dose of ABI-007 for metronomictherapy was determined by measuring the levels of circulatingendothelial progenitors (CEPs) in peripheral blood of Balb/c non-tumorbearing mice (n=5/group; dosing: 1-30 mg/kg, i.p, qd×7) with flowcytometry (Shaked et al., Cancer Cell, 7:101-111 (2005)). Subsequently,the antitumor effects of metronomic (qd; i.p.) and MTD (qd×5, 1 cycle;i.v.) ABI-007 and Taxol® were evaluated and compared in SCID micebearing human MDA-MD-231 breast and PC3 prostate cancer xenografts.

Results: ABI-007 at 5 nM significantly (p<0.05) inhibited rat aorticmicrovessel outgrowth, human endothelial cell proliferation and tubeformation by 53%, 24%, and 75%, respectively. The optimal dose ofABI-007 for metronomic therapy was observed to be 6-10 mg/kg based onCEP measurements. Metronomic ABI-007 (6 mg/kg) but not Taxol® (1.3mg/kg) significantly (p<0.05) suppressed tumor growth in both xenograftmodels. Neither ABI-007 nor Taxol® administered metronomically inducedany weight loss. Although MTD ABI-007 (30 mg/kg) inhibited tumor growthmore effectively than MTD Taxol® (13 mg/kg), significant weight loss wasnoted with the former. Interestingly, the antitumor effect of metronomicABI-007 approximated that of MTD Taxol®.

Conclusion: ABI-007 exhibits potent antiangiogenic and antitumoractivity when used in a metronomic regime.

Example 4b

Rat Aortic Ring Assay. Twelve-well tissue culture plates were coatedwith Matrigel (Collaborative Biomedical Products, Bedford, Mass.) andallowed to gel for 30 min at 37° C. and 5% CO₂. Thoracic aortas wereexcised from 8- to 10-week-old male Sprague-Dawley rats, cut into1-mm-long cross-sections, placed on Matrigel-coated wells and coveredwith an additional Matrigel. After the second layer of Matrigel had set,the rings were covered with EGM-II and incubated overnight at 37° C. and5% CO₂. EGM-II consists of endothelial cell basal medium (EBM-II;Cambrex, Walkersville, Md.) plus endothelial cell growth factorsprovided as the EGM-II Bulletkit (Cambrex). The culture medium wassubsequently changed to EBM-II supplemented with 2% FBS, 0.25 pg/mlamphotericin B and 10 pg/ml gentamycin. Aortic rings were treated withEBM-II containing the vehicle (0.9% saline/albumin),carboxyamidotriazole (CAI; 12 pg/ml), or ABI-007 (0.05-10 nM paclitaxel)for 4 days and photographed on the fifth day. CAI, a knownanti-angiogenic agent, was used at a higher than clinically achievableconcentration as a positive control. Experiments were repeated fourtimes using aortas from four different rats. The area of angiogenicsprouting, reported in square pixels, was quantified using AdobePhotoshop 6.0.

As shown in FIG. 1A, ABI-007 significantly inhibited rat aorticmicrovessel outgrowth in a concentration-dependent manner relative tothe vehicle control, reaching statistical significance (p<0.05) at 5 nM(53% inhibition) and 10 nM (68% inhibition). The amount of albuminpresent at each concentration of ABI-007 alone did not inhibitangiogenesis.

Endothelial Cell Proliferation Assay. Human umbilical vein endothelialcells (HUVEC; Cambrex) were maintained in EGM-II at 37° C. and 5% CO₂.HUVECs were seeded onto 12-well plates at a density of 30,000 cells/welland allowed to attach overnight. The culture medium was then aspirated,and fresh culture medium containing either the vehicle (0.9%saline/albumin), or ABI-007 (0.05-10 nM paclitaxel) was added to eachwell. After 48 h, cells were trypsinized and counted with a Coulter Z1counter (Coulter Corp., Hialeah, Fla.). All experiments were repeatedthree times.

As shown in FIG. 1B, human endothelial cell proliferation wassignificantly inhibited by ABI-007 at 5 nM and 10 nM by 36% and 41%,respectively.

Endothelial Cell Tube Formation Assay. Eight-well slide chambers werecoated with Matrigel and allowed to gel at 37° C. and 5% CO₂ for 30 min.HUVECs were then seeded at 30,000 cells/well in EGM-II containing eitherthe vehicle (0.9% saline/albumin) or ABI-007 (0.05-10 nM paclitaxel) andincubated at 37° C. and 5% CO₂ for 16 h. After incubation, slides werewashed in PBS, fixed in 100% methanol for 10 s, and stained withDiffQuick solution II (Dade Behring Inc., Newark, Del.) for 2 min. Toanalyze tube formation, each well was digitally photographed using a2.5× objective. A threshold level was set to mask the stained tubes. Thecorresponding area was measured as the number of pixels using MetaMorphsoftware (Universal Imaging, Downingtown, Pa.). Experiments wererepeated three times.

As shown in FIG. 1C, ABI-007 blocked tube formation by 75% at both 5 nMand 10 nM.

Determination of the In Vivo Optimal Biologic Dose of ABI-007 byMeasuring Circulating Endothelial Cells (CECs) and CirculatingEndothelial Progenitors (CEPs). Six- to eight-week-old female Balb/cJmice were randomized into the following eight groups (n=5 each):untreated, treated with i.p. bolus injections of either the drug vehicle(0.9% saline/albumin), or ABI-007 at 1, 3, 6, 10, 15 or 30 mg/kgpaclitaxel daily for 7 days. At the end of the treatment period, bloodsamples were drawn by cardiac puncture and collected in EDTA-containingvacutainer tubes (Becton Dickinson, Franklin Lakes, N.J.). CECs and CEPswere enumerated using four-color flow cytometry. Monoclonal antibodiesspecific for CD45 were used to exclude CD45+ hematopoietic cells. CECsand their CEP subset were depicted using the murine endothelial markersfetal liver kinase 1/VEGF receptor 2 (flk-1/VEGFR2), CD13, and CD117 (BDPharmingen, San Diego, Calif.). Nuclear staining (Procount; BDBiosciences, San Jose, Calif.) was performed to exclude the possibilityof platelets or cellular debris interfering with the accuracy of CEC andCEP enumeration. After red cell lysis, cell suspensions were evaluatedby a FACSCalibur (BD Biosciences) using analysis gates designed toexclude dead cells, platelets, and debris. At least 100,000events/sample were obtained in order to analyze the percentage of CECsand CEPs. The absolute number of CECs and CEPs was then calculated asthe percentage of the events collected in the CEC and CEP enumerationgates multiplied by the total white cell count. Percentages of stainedcells were determined and compared to the appropriate negative controls.Positive staining was defined as being greater than non-specificbackground staining. 7-aminoactinomycin D (7AAD) was used to enumerateviable versus apoptotic and dead cells.

FIG. 2 shows that ABI-007 administered i.p. daily for 7 days at 3, 10-30mg/kg significantly decreased CEP levels in non-tumor bearing Balb/cJmice. However, ABI-007 at 10-30 mg/kg was associated with a significantreduction of white blood cell count indicative of toxicity. Although thereduction of CEP levels by ABI-007 at 6 mg/kg did not reach statisticalsignificance, decrease in white blood cell count was not evident.Therefore it was concluded that the in vivo optimal biologic dose formetronomic ABI-007 was between 3-10 mg/kg. In one study, metronomicTaxol® at 1.3, 3, 6, or 13 mg/kg given i.p. daily for 7 days did notsignificantly reduce viable CEP levels, whereas metronomic Taxol® at 30mg/kg or higher resulted in severe toxicity and eventually mortality inmice. It was previously reported that the i.p. administration of Taxol®at doses commonly used in the clinic resulted in entrapment ofpaclitaxel in Cremophor® EL micelles in the peritoneal cavity andconsequently, insignificant plasma paclitaxel concentration (Gelderblomet al., Clin. Cancer Res. 8:1237-41 (2002)). This would explain whydoses of metronomic Taxol® (1.3, 3, 6, and 13 mg/kg) that did not causedeath failed to change viable CEP levels. In this case, the i.p.administration of metronomic Taxol® at 1.3 mg/kg would not be anydifferent from that at 13 mg/kg. Therefore the lower dose, 1.3 mg/kg,was selected to minimize the amount of Cremophor® EL per paclitaxeladministration for subsequent experiments.

Antitumor effects of metronomic and MTD ABI-007 compared with metronomicand MTD Taxol®. Human prostate cancer cell line PC3 and human breastcancer cell line MDA-MD-231 were obtained from the American Type CultureCollection (Manassas, Va.). PC3 cells (5×10⁶) were injected s.c. into 6-to 8-week-old male SCID mice, whereas MDA-MB-231 cells (2×10⁶) wereimplanted orthotopically into the mammary fat pad of female SCID mice.When the primary tumor volume reached approximately 150-200 mm³, animalswere randomized into eight groups (n=5-10/group). Each group was treatedwith either 0.9% saline/albumin vehicle control, Cremophor® EL vehiclecontrol, metronomic Taxol® (1.3 mg/kg, i.p., qd), metronomic ABI-007 (3,6, or 10 mg/kg paclitaxel, i.p., qd), MTD Taxol® (13 mg/kg, i.p., qd×5,1 cycle), or MTD ABI-007 (30 mg/kg paclitaxel, i.v., qd×5, 1 cycle).Perpendicular tumor diameters were measured with a caliper once a weekand their volumes were calculated. At the end of the treatment period,blood samples were drawn by cardiac puncture from mice in all groups.CECs and CEPs were enumerated as described herein.

Metronomic ABI-007 (3, 6 and 10 mg/kg) but not Taxol® (1.3 mg/kg)administered i.p. daily for 4 weeks significantly (p<0.05) inhibitedgrowth of both MDA-MB-231 and PC3 tumors (FIG. 3A and FIG. 3B). NeitherABI-007 nor Taxol® administered metronomically induced any weight loss(FIG. 3C and FIG. 3D). Although MTD ABI-007 (30 mg/kg) inhibited tumorgrowth more effectively than MTD Taxol® (13 mg/kg), significant weightloss was noted with the former, indicating toxicity. In addition, twoout of five mice treated with MTD ABI-007 displayed signs of paralysisin one limb 6 days after the last dose of drug. The paralysis wastransient and resolved within 24-48 hours. Interestingly, the antitumoreffect of metronomic ABI-007 at 6 mg/kg approximated that of MTD Taxol®in the MDA-MB-231 xenograft model (FIG. 3A). Increasing the dose ofmetronomic ABI-007 to 10 mg/kg did not seem to confer more pronouncedtumor growth inhibition. In contrast, metronomic ABI-007 elicitedgreater antitumor response at 10 mg/kg than at 3 and 6 mg/kg in the PC3xenografts (FIG. 3B).

Metronomic ABI-007 significantly decreased the levels of viable CEPs ina dose-dependent manner in MDA-MB-231 tumor-bearing mice (FIG. 4A).Viable CEP levels also exhibited a dose-dependent reduction in responseto metronomic ABI-007 in PC3 tumor-bearing mice, but reached statisticalsignificance only at 10 mg/kg (FIG. 4B). The levels of CEPs were notaltered by metronomic Taxol® in both xenograft models (FIGS. 4A and 4B).

Effects of metronomic and MTD ABI-007 and metronomic and MTD Taxol® onintratumoral microvessel density were studied. Five-um thick sectionsobtained from frozen MDA-MB-231 and PC3 tumors were stained with H&E forhistological examination by standard methods known to one skilled in theart. For detection of microvessels, sections were stained with a ratanti-mouse CD31/PECAM-1 antibody (1:1000, BD Pharmingen) followed by aTexas Red-conjugated goat anti-rat secondary antibody (1:200, JacksonImmunoResearch Laboratories, Inc., West Grove, Pa.). A singlemicrovessel was defined as a discrete cluster or single cell stainedpositive for CD31/PECAM-1d, and the presence of a lumen was not requiredfor scoring as a microvessel. The MVD for each tumor was expressed asthe average count of the three most densely stained fields identifiedwith a 20× objective on a Zeiss AxioVision 3.0 fluorescence microscopicimaging system. Four to five different tumors per each vehicle controlor treatment group were analyzed.

In MDA-MB-231 tumors, metronomic ABI-007 at 6 and 10 mg/kg as well asMTD ABI-007 seemed to reduce microvessel density (MVD) slightly althoughstatistical significance was not reached (FIG. 5A). In PC3 tumors,metronomic ABI-007 at 3 and 10 mg/kg appeared to decrease MVD butwithout reaching statistical significance (FIG. 5A). Interestingly, asignificant correlation existed between MVD and the level of viable CEPsin the MDA-MB-231 (FIG. 5B; r=0.76, P-0.04) but not in the PC3 (FIG. 5C;r=0.071, P-0.88) model.

In vivo angiogenesis evaluation were carried out. A Matrigel plugperfusion assay was performed with minor modifications to methods knownby one skilled in the art. Briefly, 0.5 ml Matrigel supplemented with500 ng/ml of basic fibroblast growth factor (bFGF; R&D Systems Inc.,Minneapolis, Minn.) was injected s.c. on day 0 into the flanks of10-week-old female Balb/cJ mice. On day 3, animals were randomlyassigned to eight groups (n=5 each). Each group was treated with either0.9% saline/albumin vehicle control, Cremophor® EL vehicle control,metronomic Taxol® (1.3 mg/kg, i.p., qd), metronomic ABI-007 (3, 6, or 10mg/kg paclitaxel, i.p., qd), MTD Taxol® (13 mg/kg, i.v., qd×5), or MTDABI-007 (30 mg/kg paclitaxel, i.v, qd×5). As a negative control, fiveadditional female Balb/cJ mice of similar age were injected withMatrigel alone. On day 10, all animals were injected i.v. with 0.2 ml of25 mg/ml FITC-dextran (Sigma, St. Louis, Mo.). Plasma samples weresubsequently collected. Matrigel plugs were removed, incubated withDispase (Collaborative Biomedical Products, Bedford, Mass.) overnight at37° C., and then homogenized. Fluorescence readings were obtained usinga FL600 fluorescence plate reader (Biotech Instruments, Winooski, Vt.).Angiogenic response was expressed as the ratio of Matrigel plugfluorescence to plasma fluorescence.

Metronomic ABI-007 at 6 and 10 mg/kg appeared to decrease angiogenesisalthough the inhibition did not reach statistical significance (FIG. 6).Angiogenesis seemed to be unaltered by metronomic ABI-007 at 3 mg/kg,MTD ABI-007, MTD and metronomic Taxol® relative to the respectivevehicle controls (FIG. 6). These observations were similar to theintratumoral MVD results described herein.

Example 5 Nab-5109, A Nanoparticle Albumin-Bound IDN5109 (nab-5109)Shows Improved Efficacy and Lower Toxicity Over the Tween® Formulation(Tween®-5109, Ortataxel)

Methods: Nanoparticle nab-5109 was prepared using nab technology andcharacterized by laser light scattering. Nab-5109 and Tween-5109 weretested against Pgp+DLD-1 (known to be resistant against paclitaxel anddocetaxel—Vredenburg et al, JNCI 93: 1234-1245, 2001) human coloncarcinoma xenograft in nude mice (n=5/group) at doses of 50 mg/kg(Tween®-5109, previously shown as MTD) and 75 mg/kg (nab-5109) givenq3d×4, i.v. Control groups of PBS and human serum albumin (HSA) werealso used.

Results: Nab-5109 yielded nanoparticles with mean size, Z_(Ave)=19 nmand Zeta potential=−32.7 mV. Nab-5109 was lyophilized to a dry powderthat easily dispersed in saline. In vivo, there was significantly moreweight loss (ANOVA, p<0.001) in the tumor bearing animals withTween®-5109 (50 mg/kg, 8.8% wt loss) than with nab-5109 (75 mg/kg, 3.4%wt loss) indicating substantially lower toxicity of nab-5109 despite the50% higher dose. There was significant tumor suppression by nab-5109 andTween®-5109 (ANOVA, p<0.0001 vs. controls) with tumor growth delays of36 and 28 days respectively for nab-5109 (75 mg/kg) and Tween −5109 (50mg/kg). Nab-5109 was more effective than Tween®-5109 (ANOVA, p=0.0001)in suppressing tumor growth. There were no differences between the PBSand HSA control group in term of toxicity and efficacy.

Conclusion: Nanoparticle albumin-bound, nab-5109 was successfullyprepared and could be given at 50% higher dose than Tween®-5109 withlower toxicity despite higher dose. At this higher dose, 75 mg/kg(q3d×4), nab-5109 showed significantly improved efficacy in the Pgp+DLD-1 human colon xenograft compared with Tween®-5109.

Example 6 Nanoparticle Albumin Bound (nab) Dimeric Thiocolchicinesnab-5404, nab-5800, and nab-5801: A Comparative Evaluation of AntitumorActivity vs Abraxane® and Irinotecan

Methods: Nanoparticle colchicines were prepared using nab technology.Cytotoxicity was evaluated in vitro using human MX-1 breast carcinomacultures. In vivo anti-tumor activity (human HT29 colon tumor xenograft)in nude mice was compared against Irinotecan and Abraxane®. Dose levelsfor the nab-colchicines and Irinotecan were 20 mg/kg, 30 mg/kg, and 40mg/kg, given q3d×4, i.v. Abraxane® was dosed at its MTD, 30 mg/kg, givenqd×5.

Results: The hydrophobic thiocolchicine dimers yielded nanoparticleswith average size Z_(Ave) (nm) of 119, 93, and 84 for nab-5404,nab-5800, and nab-5801, respectively. The nanoparticle suspensions weresterilized through 0.22 um filters and lyophilized. In vitro, nab-5404was the most potent of the three analogs against MX-1 (p≦0.0005, ANOVA),(IC₅₀ (ug/ml): 18, 36 and 77 for nab-5404, nab-5800 and nab-5801respectively) as well as against the HT29 xenograft in vivo (p<0.0001,ANOVA). Tumor volume was suppressed by 93%, 79%, and 48% with nab-5404at doses 40 mg/kg, 30 mg/kg, and 20 mg/kg, respectively. In contrast,tumor volume was only suppressed by 31%, 16%, and 21% with nab-5800; and17%, 30%, and 23% with nab-5801 at 40 mg/kg, 30 mg/kg, and 20 mg/kg,respectively. Nab-5404 was more effective than Irinotecan at all doselevels (p<0.008, ANOVA) with tumor volumes for Irinotecan suppressed byonly 48%, 34%, and 29% at dose levels of 40 mg/kg, 30 mg/kg, and 20mg/kg, respectively. In comparison to Abraxane , nab-5404 was moreactive at equitoxic dose (ETD) based on equal weight loss (p<0.0001,ANOVA). Tumor volume was suppressed 93% by nab-5404 (40 mg/kg, q4d×3)and 80% by Abraxane® (30 mg/kg, qd×5) at their respective ETDs.

Conclusions: Nab technology was utilized to convert 3 hydrophobicdimeric thiocolchicines (IDN5404, IDN5800, IDN5801) to nanoparticlessuitable for I.V. administration. Nab-5404 had superior antitumoractivity in vitro and in vivo compared to nab-5800 and nab-5801.Nab-5404 was more potent than Irinotecan at equal dose. At equitoxicdose, defined by weight loss, nab-5404 was more potent than Abraxane®.These data warrant further investigation of nab-5404.

Example 7 Abraxane® vs Taxotere®: A Preclinical Comparison of Toxicityand Efficacy

Methods: Toxicity of Abraxane® and Taxotere® was compared in adose-ranging study in nude mice given the drugs on a q4d×3 schedule. Thedose levels were Taxotere® 7, 15, 22, 33, and 50 mg/kg and ABX 15, 30,60, 120, and 240 mg/kg. Antitumor activity of Abraxane® and Taxotere®was compared in nude mice with human MX-1 mammary xenografts at a doseof 15 mg/kg weekly for 3 weeks.

Results: In the dose-escalation study in mice, the Taxotere® maximumtolerated dose (MTD) was 15 mg/kg and lethal dose (LD₁₀₀) was 50 mg/kg.In contrast, the Abraxane® MTD was between 120 and 240 mg/kg and LD₁₀₀was 240 mg/kg. In the tumor study Abraxane® was more effective thanequal doses of Taxotere® in tumor growth inhibition (79.8% vs 29.1%,p<0.0001, ANOVA).

Conclusion: Nanoparticle abumin-bound paclitaxel (Abraxane®) wassuperior to Taxotere® in the MX-1 tumor model when tested at equaldoses. Furthermore, the toxicity of Abraxane® was significantly lowerthan that of Taxotere®, which would allow dosing of Abraxane® atsubstantially higher levels. These results are similar to the enhancedtherapeutic index seen with Abraxane® compared to Taxol® and suggestthat the presence of surfactants may impair the transport, antitumoractivity and increase the toxicity of taxanes. Studies in additionaltumor models comparing Abraxane® and Taxotere® are ongoing.

Example 8 A Nanoparticle Albumin Bound Thiocolchicine dimer (nab-5404)with Dual Mechanisms of Action on Tubulin and Topoisomerase-1:Evaluation of In Vitro and In Vivo Activity

Methods: IDN5404 was tested for cytotoxic activity using the MCF7-Sbreast carcinoma and its multidrug resistant variant, MCF7-R (pgp+). Itscytotoxicity was also assessed against the NCI-60 human tumor cell linepanel. The nanoparticle albumin bound nab-5404 was administered IV usingvarious schedules, to SCID mice implanted s.c. with a human A121 ovariantumor xenograft.

Results: Against MCF7 cell lines, the parent compound, colchicine,demonstrated tumor growth inhibition with the IC₅₀ value (50% growthinhibitory concentration) for MCF7-S cells at 3.9±0.2 nM. The resistantvariant MCF7-R demonstrated an IC₅₀ of 66±8.6 nM, approximately a17-fold increase due to drug resistance. IDN5404, demonstrated increasedactivity against both cell lines, displaying IC₅₀ values of 1.7±0.1 and40±3.8 nM, respectively. These results were confirmed within the NCI 60human tumor cell line panel with IDN5404 having a mean IC₅₀ of <10⁻⁸Mand >10 fold resistance between the MCF7-S and the MCF7-R cell lines.The COMPARE algorithm identified IDN5404 as a tubulin binder similar tovinca alkaloids, confirming the previous results. In vivo against theA121 ovarian tumor xenograft, efficacy and toxicity of nab-5404 was doseand schedule dependent. Nanoparticle nab-5404 was well tolerated andcapable of inducing complete regressions and cures: at 24 mg/kgadministered IV qd×5, 5 of 5 mice were long-term survivors (LTS) with noevidence of tumor. However, increasing the dosage to 30 mg/kg resultedin 5 of 5 toxic deaths. On a q3d×4 schedule, 30 mg/kg resulted in 4 of 5mice LTS and at 50 mg/kg, 5 of 5 toxic deaths. Using a q7d×3 schedule,40 mg/kg resulted in 3 of 5 mice LTS and at 50 mg/kg, 4 of 4 LTS werenoted.

Conclusions: IDN5404, a new thiocolchicine dimer with dual mechanism ofaction showed activity in pgp-expressing, cisplatin and topotecanresistant cell lines. In vivo, nanoparticle albumin bound nab-5404 wasactive against A121 ovarian xenografts.

Example 9 Combination Studies of Abraxane® and Other Agents

Due to the advantageous properties of Abraxane® (ABX, the nanoparticlealbumin-bound paclitaxel) noted above, it was used and being used in anumber of studies with different modes of administration and schedulesand in combination with other oncology drugs as well as radiationtreatment. These are listed below:

In metastatic breast cancer, these studies include:

Randomized Phase II Trial of ABX 125, Gem 1000 mg/m², To evaluate thecombination of ABX Weekly Abraxane ® in Combination D1, 8; q 3wk andGemcitabine in 1st-line MBC. with Gemcitabine in Individuals with HER2Negative Metastatic Breast Cancer A phase II study of weekly ABX 100mg/m², Carbo AUC Data will be important for using dose-densenanoparticle paclitaxel 2, both D1, 8, 15; Her 2 mg/kg ABX incombination with carbo (ABI-007) carboplatin, with (4 mg/kg on wk a)q4wk × 6 and/or Herceptin ®. Also helpful for Herceptin ® as first orsecond-line other combinations. therapy of advanced HER2 positive breastcancer Weekly Vinorelbine and Abraxane ®, L1: ABX 80, Nav 15; L2:Multi-center study of ABX in with or without G-CSF, in stage IV ABX 90,Nav 20; L3: ABX combination with Navelbine ® in breast cancer: a phaseI-II study 100, Nav 22.5; L4: ABX 110, 1st-line MBC. Nav 25; L5: ABX125, Nav 25 qwk Phase II trial of weekly Abraxane ® ABX 125 mg/m² Q3/4wkA relatively large phase II of weekly monotherapy for 1st-line MBC (plusABX monotherapy at 125 mg/m² in Herceptin ® in Her2+ pts) 1st-line MBC.Phase I/II trial Abraxane ® plus ABX + Anthracycline Doxil ® for MBCplus limited PK 3-arm phase II trial in 1st-line MBC ABX weekly (130mg/m²) vs. To optimize ABX monotherapy q2wk (260 mg/m²) vs. q3wk regimefor MBC (260 mg/m²) 3-arm phase II trial in 1st-line and ABX weekly vs.ABX q3wk randomized ABX MBC trial to 2nd-line MBC, with biological vs.Taxol ® weekly obtain important data: weekly ABX correlates analyses vs.weekly Taxol ®; weekly ABX vs. 3-weekly ABX; plus biomarker study(caveolin-1 and SPARC). Phase I/II Abraxane ® + GW572016 TBD combinationof ABX and GW572016 (a dual EGFR inhibitor and one of the most promisingnew biological agents for BC). A phase I dose escalation study of aAbraxane ® 100 mg/m² weekly, This phase I trial is to determine the 2day oral gefitinib 3 out of 4 weeks; Gefitinib safety and tolerabilityof a 2 day chemosensitization pulse given prior starting at 1000 mg/d ×2 days gefitinib pulse given prior to to weekly Abraxane ® inindividuals Abraxane ® administration. with advanced solid tumors PhaseII 1^(st) line MBC trial weekly ABX (125 mg/m², 2 To evaluate thecombination of ABX wk on and 1 wk off) + Xeloda ® and Xeloda ® in1st-line MBC, using 825 mg/m² d 1-14 q3wk 2 weekly on and 1 weekly offABX regime. Phase II pilot adjuvant trial of Dose dense AC + G CSF --> Apilot adjuvant study of a “super Abraxane ® in breast cancer weekly ABX--> Avastin ® dose dense” Abraxane ® in dose-dense adjuvant AC q2w × 4 +G CSF --> ABX A pilot adjuvant study of dose dense chemotherapy forearly stage breast q2wk × 4 ABX regime -- an alternate of a cancerstandard adjuvant regime Phase II pilot adjuvant trial of AC Q2wk -->ABX q2wk + A pilot adjuvant study in preparation Abraxane ® in breastcancer G-CSF for phase III adjuvant trial

In Breast cancer neoadjuvant setting studies Include:

Phase II Trial of Dose Dense Neoadjuvant: Gem 2000, This neoadjuvantstudy is based on the Neoadjuvant Gemcitabine, Epirubicin, Epi 60, ABX175 mg/m², GET data from Europe which showed ABI-007 (GEA) in LocallyAdvanced Neul 6 mg SC, all D1 q2wk × high activity. In the currentregime, or Inflammatory Breast Cancer 6 Adjuvant: Gem ABX will replaceT, or Taxol ®. 2000, ABX 220, Neul 6 mg D1 q2wk × 4 Phase IIpreoperative trial of ABX 220 mg/m² q2wk × Abraxane ® followed by FEC (+6 followed by FEC × 4 Herceptin ® as appropriate) in breast(+Herceptin ® for Her2+ cancer pts) Pre-clinical study of drug-drugABX + other agents interaction Phase II neoadjuvant (ABX + Herceptin ®)followed by (Navelbine ® + Herceptin ®) Randomized phase II trial of TACvs. AC followed To evaluate AC followed by neoadjuvant chemotherapy inABX + carbo vs. AC ABX/carbo or ABX/carbo/Herceptin ® individuals withbreast cancer followed combinations vs TAC (a FDA ABX + carbo +Herceptin ® approved adjuvant BC regime) in neoadjuvant setting. PhaseII neoadjuvant trial of ABX: 200 mg/m² D1; Abraxane ® and capecitabinein locally Xel: 1000 mg/m² D1-14; advanced breast cancer q3wk × 4 PhaseII trial of neoadjuvant ABX: 300 mg/m² q3wk chemotherapy (NCT) withnanoparticle paclitaxel (ABI-007, Abraxane ®) in women with clinicalstage IIA, IIB, IIIA, IIIB, and IV (with intact primary) breast cancers

In lung cancer the studies include:

Phase I/II study of Abraxane ® ABX weekly The first phase II trial ofABX monotherapy in 1st-line advanced combo with carbo in NSCLC. NSCLCPhase II Trial of weekly Abraxane ® ABX: 125 mg/m² plus carboplatin in1st-line NSCLC D1, 8, 15; Carbo: AUC 6 D1; q4 wk A Phase I Trial ofCarboplatin and Arm 1: ABX 100, 125, This 2-arm phase I study willAbraxane ® on a weekly and every 150 mg/m² D1, 8, 15 generate importantdata on three week schedule in individuals q4wk; Arm 2: ABX ABX/carbocombination for with Advanced Solid Tumor 220, 260, 300 mg/m² furtherstudies of this combo Malignancies D1 q3wk. Carbo AUC6 in multiplediseases. in both arms Phase II study of ABI 007 ABX Level(a): 225mg/m2; This phase II NSCLC study (Abraxane ®) and carboplatin inLevel(b): 260 mg/m2; will generate data for a future advanced non-smallcell lung cancer. Level(3): 300 mg/m2; phase III registration trial inq3wk Carbo lung cancer fixed at AUC6 q3wk Phase I study of ABI 007(Abraxane ®) ABX q3wk and carboplatin Phase I/II study of Abraxane ® +TBD ABX and Alimta ® can be a Alimta ® for 2nd-line NSCLC promisingcombination due to the non-overlapping toxicity profiles. Phase I/IItrial of Abraxane ® plus cisplatin in advanced NSCLC Phase I/II study ofAbraxane ®, Navelbine ®, and Cisplatin for treatment of advanced NSCLCPhase II ABX mono in 1st-line ABX 260 mg/m² q3wk The 1st ABX trial inNSCLC. NSCLC Phase II study of Abraxane ® Cohort 1: ABX q3wk;monotherapy in 2nd-line NSCLC Cohort 2: ABX weekly. Doses TBD Phase I/IItrial of weekly Abraxane ® 1st line and carboplatin in advanced NSCLC

Studies in Prostate include:

Randomized phase II ABX 100 mg/m² weekly vs Q3W in front line weekly vs260 mg/m² HRP q3wk Phase II ABX in 1st-line weekly ABX Phase II study ofweekly ABX in 1st-line prostate cancer HRPC Phase II neoadjuvant studyTBD A multi-center neoadjuvant trial of ABX in prostate cancer plusbiomarker study. Phase II ABX 100 mg weekly no break

Studies in ovarian cancer include:

Phase II study of Abraxane ® for TBD treatment of advanced ovariancancer (3rd-line) Phase I study of Abraxane ® plus ABX weekly + Carbocarbo for treatment of advanced AUC 6 ovarian cancer A phase II trial ofAbraxane ®/Carboplatin in recurrent ovarian cancer

Studies in Chemoradiation include:

Phase I/II trial of Abraxane ® combined with radiation in NSCLCAbraxane ® Combined With animal model Radiation H&N (Head and NeckCancer) TBD

Other studies include:

Phase II study of ABX in treatment of 125 mg/m² d1, 8, 15 persistent orrecurrent carcinoma of the q28 days cervix PhII in preciously treated(100 ABX) 26-->70 and untreated (150 ABX) metastatic melanoma Ph IIsingle treatment use of ABI-007 for the treatment of non-hematologicmalignancies Abraxane ® Combined With antiangiogenic agents, e.g.,Avastin ®. Abraxane ® Combined With proteasome inhibitors e.g.,Velcade ®. Abraxane ® Combined With EGFR inhibitors e.g., Tarceva ®. Arandomized phase II trial of weekly gemcitabine, Abraxane ®, andexternal irradiation for locally advanced pancreatic cancer

Example 10 Combination of Nanoparticle Invention Drugs with Other Agentsand Modes of Therapy

Lower toxicity of nanoparticle invention drugs described herein allowcombination with other oncology drugs and other modes of treatment withmore advantageous outcome. These include nanoparticle forms ofpaclitaxel, docetaxel, other taxanes and analogs, geldanamycins,colchicines and analogs, combretastatins and analogs, hydrophobicpyrimidine compounds, lomaiviticins and analogs including compounds withthe lomaiviticin core structures, epothilones and analogs,discodermolide and analogs and the like. The invention drugs may becombined with paclitaxel, docetaxel, carboplatin, cisplatin, otherplatins, doxorubicin, epirubicin, cyclophosphamide, iphosphamide,gemcitabine, capecitabine, vinorelbine, topotecan, irinotecan,tamoxifen, camptothecins, 5-FU, EMP, etoposide, methotraxate and thelike.

Example 11 Combination of Abraxane® with Carboplatin and Herceptin®

The combination of Taxol® and carboplatin has shown significant efficacyagainst metastatic breast cancer. On a weekly schedule, in thiscombination, Taxol® can only be dosed at up to 80 mg/m². Higher dosescannot be tolerated due to toxicity. In addition, HER-2-positiveindividuals derive greater benefit when Herceptin® is included in theirtherapeutic regime. This open-label Phase II study was conducted todetermine the synergistic therapeutic effect of ABI-007 (Abraxane®) withthese agents. The current study was initiated to evaluate the safety andantitumor activity of ABI-007/carboplatin with Herceptin® forindividuals with HER-2 positive disease. ABI-007 was given incombination with carboplatin and Herceptin® administered intravenouslyweekly to individuals with HER-2 positive advanced breast cancer. Acohort of 3 individuals received ABI-007 at a dose of 75 mg/m² IVfollowed by carboplatin at target AUC=2 weekly and Herceptin® infusion(4 mg/kg at week 1, and 2 mg/kg on all subsequent weeks) for 1 cycle.These individuals tolerated the drug very well so for all subsequentcycles and individuals the dose of ABI-007 was escalated to 100 mg/m².Six individuals were treated to date. Of the 4 individuals that wereevaluated for response, all 4 (100%) showed a response to the therapy.It should be noted that due to lower toxicity of Abraxane®, a highertotal paclitaxel dose could be given compared to Taxol® with resultingbenefits to the individuals.

Example 12 Combination of Abraxane® with Carboplatin

The combination of Taxol® and carboplatin has shown significant efficacyin lung cancer. Another study with Abraxane® in combination withcarboplatin on a 3 weekly schedule in individuals with lung cancer isongoing.

Example 13 Use of Abraxane® in Combination with Radiation Example 13a

Abraxane®, combined with clinical radiotherapy, enhances therapeuticefficacy and reduces normal tissue toxicity. Abraxane® is used toincrease the therapeutic gain of radiotherapy for tumors; to enhancetumor response to single and fractionated irradiation; to enhance normaltissue response to radiation and to increase therapeutic ratio ofradiotherapy.

A murine ovarian carcinoma, designated OCa-I, which has beeninvestigated extensively is used. First, optimal timing of Abraxane®administration relative to local tumor radiation is timed to producemaximum antitumor efficacy. Tumors are generated in the right hind legof mice by i.m. injection of tumor cells and treatment is initiated whenthe tumors reach 8 mm in size. Mice are treated with 10 Gy single doseirradiation, a single dose of Abraxane® or with combination therapy ofAbraxane® given at different times 5 days before to 1 day afterirradiation. A dose of Abraxane® equal to about 1½ times more than themaximum tolerated dose of paclitaxel is used, a dose of 90 mg/kg. Theendpoint of efficacy is tumor growth delay. The groups consist of 8 miceeach. Tumors are generated and treated as described in Aim 1. Theendpoint of efficacy is tumor growth delay. Tumors are irradiated with5, 7.5 or 10 Gy delivered either in a single dose or in fractionateddoses of 1, 1.5 or 2 Gy radiation daily for five consecutive days. SinceAbraxane® is retained in the tumor for several days and exerts itsenhancing effect on each of the five daily fractions, Abraxane® is givenonce at the beginning of the radiation regime. Since the ultimate goalin clinical radiotherapy is to achieve tumor cure, the potential forAbraxane to enhance tumor radiocurability is determined. The same schemeas described for the fractionated tumor growth delay study is used,except that a range of doses from 2 to 16 Gy is given daily for fiveconsecutive days (total radiation dose 10 to 80 Gy). Tumors are followedfor regression and regrowth for up to 120 days after irradiation, whenTCD50 (the dose of radiation needed to yield local tumor cure in 50percent of animals) is determined. There are two TCD50 assays: radiationonly and Abraxane® plus radiation, and each assay consists of 10radiation dose groups containing 15 mice each. To provide therapeuticgain, any radioenhancing agent, including Abraxane®, must increase tumorradioresponse more than increase normal tissue damage by radiation.Damage to jejunal mucosa, a highly proliferative tissue affected bytaxanes is assessed. The jejunal microcolony assay is used to determinethe survival of crypt epithelial cells in the jejunum of mice exposed toradiation. Mice are exposed to whole body irradiation (WBI) with dailydoses of X-rays ranging from 3 to 7 Gy for five consecutive days. Themice are treated with Abraxane®, at an equivalent paclitaxel dose of 80mg/kg, administered i.v. 24 h before the first dose of WBI and killed3.5 days after the last dose of WBI. The jejunum is prepared forhistological examination, and the number of regenerating crypts in thejejunal cross-section is counted. To construct radiation survivalcurves, the number of regenerating crypts is converted to the number ofsurviving cells.

Example 13b

The objective of this study was to assess whether ABI-007 (a) as asingle agent has antitumor activity against the syngeneic murine ovariancarcinoma OCa-1 and (b) enhances the radiation response of OCa-1 tumorsin a combined treatment regime as described in the previous example withthe following modifications.

OCa-1 tumor cells were injected i.m. into the hind leg of C3H mice. Whentumors grew to a mean diameter of 7 mm, single treatment with localradiation (10 Gy) to the tumor-bearing leg, ABI-007 90 mg/kg i.v., orboth, was initiated. To determine the optimal treatment scheduling,ABI-007 was given from 5 days to 9 hours before radiation as well as 24hours after radiation. Treatment endpoint was absolute tumor growthdelay (AGD), defined as the difference in days to grow from 7-12 mm indiameter between treated and untreated tumors. For groups treated withthe combination of ABI-007 and radiation, an enhancement factor (EF) wascalculated as the ratio of the difference in days to grow from 7 to 12mm between the tumors treated with the combination and those treatedwith ABI-007 alone to the AGD of tumors treated with radiation only. Toassess the radiation-enhancing effect of ABI-007 for a fractionatedradiation regime on the endpoint tumor cure, a TCD50 assay was performedand analyzed 140 days post treatment. Total doses of 5 to 80 Gy in 5daily fractions were administered either alone or combined with ABI-00724 hours before the first radiation dose.

As a single agent, ABI-007 significantly prolonged the growth delay ofthe OCa-1 tumor (37 days) compared to 16 days for untreated tumors.ABI-007 as a single agent was more effective than a single dose of 10Gy, which resulted in a delay of 29 days. For combined treatmentregimes, ABI-007 given at any time up to 5 days before radiation,produced a supra-additive antitumor effect. EF was 1.3, 1.4, 2.4, 2.3,1.9, and 1.6 at intertreatment intervals of 9h, 24 h and 2, 3, 4, and 5days, respectively. When ABI-007 was given after radiation, the combinedantitumor treatment effect was less than additive. Combined treatmentwith ABI-007 and radiation also had a significant effect on tumor cureby shifting the TCD50 of 55.3 Gy for tumors treated with radiation onlyto 43.9 Gy for those treated with the combination (EF 1.3).

This experiment demonstrated that ABI-007 possesses single-agentantitumor activity against OCa-1 and enhances the effect of radiotherapywhen given several days prior. As previously demonstrated for paclitaxeland docetaxel, the radiation enhancement is likely a result of multiplemechanisms, with a cell cycle arrest in G2/M being dominant at shorttreatment intervals and tumor reoxygenation at longer intervals.

Example 14 Combination of Abraxane® and Tyrosine Kinase Inhibitors

Pulse-dosing of gefitinib in combination with the use of Abraxane® isuseful to inhibit the proliferation of EGFr expressing tumors. 120 nudemice are inoculated with BT474 tumor cells to obtain at least 90 micebearing BT474 xenograft tumors and split into 18 experimental arms (5mice each). Arm 1 mice receive control i.v. injections. All other micereceive weekly i.v. injections of Abraxane® at 50 mg/kg for 3 weeks. Arm2 receives Abraxane® alone. Arms 3, 4, 5, 6, 7, 8 receive weeklyAbraxane® preceded by 2 days of a gefitinib pulse at increasing doses.Arms 9, 10, 11, 12, 13 receive weekly Abraxane® preceded by one day of agefitinib pulse at increasing doses. Arms 14, 15, 16, 17, 18 receiveweekly Abraxane® along with everyday administration of gefitinib atincreasing doses. The maximum tolerated dose of gefitinib that can begiven in a 1 or 2 day pulse preceding weekly Abraxane® or in continuousadministration with Abraxane® is established. In addition, measurementof anti-tumor responses will determine whether a dose-responserelationship exists and whether 2 day pulsing or 1 day pulsing issuperior. These data are used to select the optimal dose of pulsegefitinib and that of continuous daily gefitinib given with Abraxane®

120 nude mice are inoculated with BT474 tumor cells to obtain 90 micebearing tumors. These mice are split into 6 groups (15 each). Arm 1receive control i.v. injections. Arm 2 receives Abraxane® 50 mg/kg i.v.weekly for 3 weeks. Arm 3 receive oral gefitinib at 150 mg/kg/day. Arm 4receive Abraxane® 50 mg/kg along with daily gefitinib at the previouslyestablished dose. Arm 5 receive Abraxane® 50 mg/kg preceded by agefitinib pulse at the previously established dose and duration. Arm 6receive only a weekly gefitinib pulse at the previously establisheddose. After three weeks of therapy, mice are followed until controlsreach maximum allowed tumor sizes.

Example 15 Phase II Study of Weekly, Dose-Dense Nab™-Paclitaxel(Abraxane®), Carboplatin With Trastuzumab® as First-line Therapy ofAdvanced HER-2 Positive Breast Cancer

This study aimed to evaluate (1) the safety and tolerability and (2) theobjective response rate of weekly dose-densetrastuzumab/Abraxane®/carboplatin as first-line cytotoxic therapy forpatients with advanced/metastatic (Stage 1V adenocarcinoma)HER-2-overexpressing breast cancer. Trastuzumab is a monoclonalantibody, also known as Herceptin®, which binds to the extracellularsegment of the erbB2 receptor.

Briefly, patients without recent cytotoxic or radiotherapy wereincluded. Doses of Abraxane were escalated from 75 mg/m² as 30-min i.v.infusions on days 1, 8, 15 up to 100 mg/m² for subsequent cyclesaccording to the standard 3+3 rule. Carboplatin AUC=2 was given as 30-60min i.v. infusions on days 1, 8, 15 and for an initial 29 day cycle.Trastuzumab was given as i.v. 30-90 min infusion on days 1, 8, 15, 22 ata dose of 4 mg/kg at week 1 and 2 mg/kg on all subsequent weeks.

Of 8 out of 9 patients evaluable for response the response rate(confirmed plus unconfirmed) was 63% with 38% stable disease. The mostcommon toxicities were neutropenia (grade 3: 44%; grade 4: 11%) andleukocytopenia (33%).

These results suggest that trastuzumab plus Abraxane® plus carboplatindemonstrated a high degree of antitumor activity with acceptabletolerability as a first-line therapy for MBC.

Example 16 Phase II Trial of Capecitabine Plus nab -Paclitaxel(Abraxane®) in the First Line Treatment of Metastatic Breast Cancer

The purpose of this phase II study was to evaluate the safety, efficacy(time to progression and overall survival), and quality of life ofpatients with MBC who received capecitabine in combination withAbraxane®. Capecitabine is a fluoropyrimidine carbamate also known asXeloda® which has been shown to have substantial efficacy alone and incombination with taxanes in the treatment of MBC.

In this open-label, single-arm study, Abraxane® 125 mg/m² was given byi.v. infusion on day 1 and day 8 every 3 weeks plus capecitabine 825mg/m² given orally twice daily on days 1 to 14 every 3 weeks. Patientswere HER-2/neu negative with a life expectancy of greater than 3 months.Patients had no prior chemotherapy for metastatic disease, no priorcapecitabine therapy, and no prior fluoropyrimidine therapy andpaclitaxel chemotherapy given in an adjuvant setting.

12 patients have been enrolled with safety analysis completed on thefirst 6 patients and the response rate evaluable after 2 cycles in thefirst 8 patients. There were no unique or unexpected toxicities with nograde 4 toxicities or neuropathy greater than grade 1. Response datawere confirmed on only the first 2 cycles of therapy (first evaluationpoint) in 6 patients. Two patients have completed 6 cycles with 1partial response and 1 stable disease. Of the first 8 patients after 2cycles, there were 2 partial responses and 4 with stable disease.

These results show that combination of capecitabine and weekly Abraxane®at effective doses is feasible with no novel toxicities to date.Abraxane® related toxicity was mainly neutropenia without clinicalconsequences, and hand foot syndrome was the major toxicity ofcapecitabine.

Example 17 Pilot Study of Dose-Dense Doxorubicin Plus CyclophosphamideFollowed by Nab-Paclitaxel (Abraxane®) in Patients with Early-StageBreast Cancer

The objective of this study was to evaluate the toxicity of doxorubicin(adriamycin) plus cyclophosphamide followed by Abraxane® in early stagebreast cancer.

Patients had operable, histologically confirmed breast adenocarcinoma ofan early stage. The patients received doxorubicin (adriamycin) 60 mg/m²plus cyclophosphamide 600 mg/m² (AC) every 2 weeks for 4 cycles followedby Abraxane® 260 mg/m² every two weeks for 4 cycles.

30 patients received 4 cycles of AC, and 27 of 29 patients received 4cycles of Abraxane®; 33% of patients received pegfilgrastim (Neulasta®)for lack of recovery of ANC (absolute neutrophil count) duringAbraxane®. Nine patients (31%) had Abraxane® dose reductions due tonon-hematologic toxicity. A total of 9 patients had grade 2 and 4patients had grade 3 peripheral neuropathy (PN); PN improved by >1 gradewithin a median of 28 days.

These results indicate that dose-dense therapy with doxorubicin (60mg/m²) plus cyclophosphamide (600 mg/m²) every 2 weeks for 4 cyclesfollowed by dose-dense Abraxane® (260 mg/m²) every 2 weeks for 4 cycleswas well tolerated in patients with early-stage breast cancer.

Example 18 Weekly Nab-Paclitaxel (Abraxane®) as First Line Treatment ofMetastatic Breast Cancer with Trastuzumab Add On for HER-2/Neu-PositivePatients

The purpose of the current study was to move weekly Abraxane® to afront-line setting and add trastuzumab for HER2/neu-positive patients.

This phase II, open-label study included 20 HER2-positive and 50HER2-negative patients with locally advanced or metastatic breastcancer. Abraxane® was given at 125 mg/m² by 30 minute i.v. infusion ondays 1, 8, and 15 followed by a week of rest. Trastuzumab was givenconcurrently with study treatment for patients who were HER2-positive.The primary endpoint was response rate and the secondary endpoints weretime to progression (TTP), overall survival (OS), and toxicity.

In the safety population, 23 patients received a median of 3 cycles ofAbraxane® to date. The most common treatment-related adverse event wasgrade 3 neutropenia (8.7%) with no grade 4 adverse events. One out of 4evaluable patients responded to therapy.

Example 19 Phase I Trial of Nab-Paclitaxel (Abraxane®) and Carboplatin

The aim of the current study was to determine the maximum tolerated doseof Abraxane® (both weekly and every 3 weeks) with carboplatin AUC=6 andto compare the effects of sequence of administration on pharmacokinetics(PK).

Patients with histologically or cytologically documented malignancy thatprogressed after “standard therapy” were included. Arm 1 receivedAbraxane® every 3 weeks in a dose escalation format based on cycle 1toxicities (220, 260, 300, 340 mg/m²) every 3 weeks followed bycarboplatin AUC=6. Arm 2 received weekly (days 1, 8, 15 followed by 1week off) Abraxane® (100, 125, 150 mg/m²) followed by carboplatin AUC=6.For the PK portion of the study, Abraxane® was followed by carboplatinin cycle 1 and the order of administration reversed in cycle 2 with PKlevels determined at initial 6, 24, 48 and 72 hours.

On the every 3 weeks schedule, neutropenia, thrombocytopenia andneuropathy were the most common grade ¾ toxicities ( 3/17 each). On theweekly schedule, neutropenia 5/13 was the most common grade ¾ toxicity.The best responses to weekly administration at the highest dose of 125mg/m² (n=6) were 2 partial responses (pancreatic cancer, melanoma) and 2stable disease (NSCLC). The best responses to the every three weekadministration at the highest dose of 340 mg/m² (n=5) were 1 stabledisease (NSCLC) and 2 partial responses (SCLC, esophageal).

These data indicate activity of combination of Abraxane® andcarboplatin. The MTD for the weekly administration was 300 mg/m², andfor the once every 3 week administration was 100 mg/m².

Example 20 Phase II Trial of Dose-Dense Gemcitabine, Epirubicin, andNab-Paclitaxel (Abraxane®) (GEA) in Locally Advanced/Inflammatory BreastCancer

In an open-label, phase II study an induction/neoadjuvant therapy regimewas instituted prior to local intervention. The therapy regime wasgemcitabine 2000 mg/m² i.v. every 2 weeks for 6 cycles, epirubicin 50mg/m² every 2 weeks for 6 cycles, Abraxane® 175 mg/m² every 2 weeks for6 cycles, with pegfilgrastim 6 mg s.c. on day 2 every 2 weeks. Thepostoperative/adjuvant therapy regime after local intervention wasgemcitabine 2000 mg/m² every 2 weeks for 4 cycles, Abraxane® 220 mg/m²every 2 weeks for 4 cycles and pegfilgrastim 6 mg s.c. day every 2weeks. Patients included females with histologically confirmed locallyadvanced/inflammatory adenocarcinoma of the breast.

Example 21 Cytotoxic Activity of Nab-Rapamycin in Combination withAbraxane® on Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSMC) were seeded onto 96 wells plates inthe presence of increasing concentrations of nab-rapamycin and 0 μM, 1μM, 10 μM, or 100 μM of Abraxane® (ABI-007). To evaluate the cytotoxiceffect of nab-rapamycin and Abraxane®, treated VSMCs were stained withethidium homodimer-1 (Invitrogen, Carlsbad Calif.) and analyzed for redfluorescence. Ethidium homodimer-1 is a high-affinity, fluorescentnucleic acid stain that is only able to pass through compromisedmembranes of dead cells to stain nucleic acids. As shown in FIG. 7A,nab-rapamycin, by itself, exhibited dose-dependent cell killing asdemonstrated by increasing fluorescence. Cell killing by nab-rapamycinwas not enhanced by Abraxane® at 1 μM or 10 μM; however, it was greatlyenhanced by Abraxane® at 100 μM (ANOVA, p<0.0001). Cells stained withethidium homodimer-1 as shown in FIG. 7A were also exposed to calcein.Calcein AM (Invitrogen) is a non-fluorescent molecule that is hydrolyzedinto fluorescent calcein by nonspecific cytosolic esterases. Live cellsexposed to calcein AM exhibit bright green fluorescence as they are ableto generate the fluorescent product and retain it. As shown in FIG. 7B,nab-rapamycin exhibited dose dependent cytotoxic activity as shown by areduced amount of fluorescent staining by calcein. This reduction influorescence was enhanced by coincubation with Abraxane® in a dosedependent manner. ANOVA statistic gave p<0.0001 at all drugconcentrations of Abraxane®.

Example 22 Cytotoxic Activity of Nab-Rapamycin in Combination withAbraxane® Against HT29 (Human Colon Carcinoma) Tumor Xenograft

Nude mice were implanted with 106 HT29 cells on their right flanks.Treatment was initiated once the tumor were palpable and were greaterthan 100-200 mm³. The mice were randomly sorted into 4 groups (n=8 pergroup). Group 1 received saline 3 times weekly for 4 weeks, i.v.; Group2 received Abraxane® at 10 mg/kg, daily for 5 days, i.p.; Group 3received nab-rapamycin at 40 mg/kg, 3 times weekly for 4 weeks, i.v.;and Group 4 received both nab-rapamycin (40 mg/kg, 3 times weekly for 4weeks, i.v.) and Abraxane® (10 mg/kg, daily for 5 days, i.p.). As shownin FIG. 8, the tumor suppression was greater for the Abraxane® plusnab-rapamycin combination therapy than for either single therapy group.

Example 23 Cytotoxic Activity of Nab-17-AAG in Combination withAbraxane® Against H358 (Human Lung Carcinoma) Tumor Xenograft

Nude mice were implanted with 107H358 cells on their right flanks.Treatment was initiated once the tumors were palpable and were greaterthan 100-200 mm³. The mice were randomly sorted into 4 groups (n=8 pergroup). Group 1 received saline 3 times weekly for 4 weeks, i.v.; Group2 received Abraxane® at 10 mg/kg, daily for 5 days, i.p.; Group 3received nab-17-AAG at 80 mg/kg, 3 times weekly for 4 weeks, i.v.; andGroup 4 received both nab-17-AAG (80 mg/kg, 3 times weekly for 4 weeks,i.v.) and Abraxane® (10 mg/kg, daily for 5 days, i.p.). As shown in FIG.9, the tumor suppression was greater for the nab-17-AAG plus Abraxane®combination therapy than for either single therapy group.

Example 24 Abraxane® (ABI-007) Reduces Tumor Growth in MDA-MB-231 HumanTumor Xenografts and Induces Necrosis, Hypoxia and VEGF-A Expression

MDA-MB-231 human breast cancer xenografts were orthotopically implantedin the mammary fat pads of female nude (nu/nu) mice. When the averagetumor volume was 230 mm³, mice were randomized into groups of fiveanimals and treated with saline, Taxol®, Abraxane® or doxorubicin.Taxol® was administered at 10 mg/kg/day, Abraxane® was administered at15 mg/kg/day, and doxorubicin was administered at 10 mg/kg/day. Alldrugs and control saline were administered i.v. in a volume of 100 μldaily for 5 days. Mice were sacrificed, tumors were harvested and tumorcellular extracts were prepared. VEGF-A protein levels in tumor extractswere determined by ELISA. In some cases, tumors from mice treated withAbraxane® were analyzed by histology.

TABLE 4 Mean VEGF-A Dose Tumor Volume (pg/mg Treatment Schedule (mm³) %TGI protein) Saline control 100 μl 523 ± 79 337 ± 51 qd × 5 Taxol ® 10mg/kg/day 231 ± 32 56 664 ± 66 qd × 5 Abraxane ® 15 mg/kg/day 187 ± 2964 890 ± 82 qd × 5 Doxorubicin 10 mg/kg/day 287 ± 56 45 754 ± 49 qd × 5

As shown in Table 4, Taxol®, Abraxane® and doxorubicin all inhibitedtumor growth as represented by a reduction in tumor volume when comparedto saline-treated control animals. Tumor growth inhibition (TGI) wascalculated by comparing mean tumor volume of test groups to that of thecontrol group at the last measurement of the control group. Tumor growthinhibition was greatest in mice treated with Abraxane® (64% inhibition).Taxol® and doxorubicin showed tumor growth inhibition of 56% and 45%,respectively.

Levels of VEGF-A protein in tumor cellular extracts were measured byELISA and shown to be increased in tumors from mice treated with Taxol®,Abraxane® and doxorubicin. VEGF-A protein levels were highest in tumorsfrom mice treated with Abraxane® (164% increase), followed bydoxorubicin (124%) and Taxol® (97%).

Tumors were harvested from saline-treated control mice andAbraxane®-treated mice one week after the last injection of Abraxane®.The tumors were evaluated for sites of necrosis and for the presence ofhypoxic cells. Hypoxic cells were identified by immunohistochemicaldetection of pimonidazole-protein conjugates. As shown in FIG. 10,inhibition of tumor growth in Abraxane -treated mice was accompanied bynecrosis (FIG. 10B) and hypoxia (FIG. 10D) in the tumor tissue. Necrosisand hypoxia was not observed in tumor tissue from saline-treated controlmice (FIG. 10A and FIG. 10C).

Example 25 VEGF-A and Avastin® Effects on Abraxane®-Induced In VitroCytotoxicity

In addition to the stimulation of tumor angiogenesis by acting onvascular endothelial cells, it is possible that the secreted VEGF-Acould also act on tumor cells exhibiting VEGF-A receptors (VEGF-R).MDA-MB-231 cells expressed VEGF-R2, whereas HepG2 cells expressedVEGF-R1 and PC3 prostate tumor cells expressed both VEGF-R1 and VEGF-R2(data not shown).

The effect of VEGF-A or an anti-VEGF antibody (Avastin®) onAbraxane®-induced cytotoxicity was assessed in an in vitro cellularcytotoxicity assay. Cells were treated with Abraxane® in a range ofconcentrations (1 to 24 nM). Cells were also treated with VEGF-A orAvastin® and cytotoxicity was compared to cells treated with Abraxane®alone. As shown in FIG. 11A, addition of VEGF-A reduced the in vitrocytotoxicity of Abraxane®. In contrast, the addition of Avastin®increased the in vitro cytotoxicity of Abraxane® (FIG. 11A).

Similar results were observed in an in vitro clonogenic assay. Cellswere treated with saline control, Abraxane® alone, VEGF-A alone,Avastin® alone, Abraxane®+VEGF-A or Abraxane®+Avastin®. As shown in FIG.11B, Abraxane® reduced the mean number of colonies formed as compared tosaline control. Treatment with VEGF-2 alone increased the number ofcolonies formed, while treatment with Avastin® alone resulted in aslight reduction in the number of colonies formed. The addition ofVEGF-A to Abraxane®-treated cells reduced the cytotoxic effect whichresulted in a higher number of colonies formed as compared to Abraxane®alone. The addition of Avastin® to Abraxane®-treated cells appeared tohave a synergistic effect demonstrating an increase in cytotoxicity (asdemonstrated by a sharp decrease in number of colonies formed) over thelevel observed with Abraxane® or Avastin® alone.

Example 26 Abraxane® (ABI-007) in Combination with Avastin® ReducesTumor Growth in MDA-MB-231 Tumor Xenografts

Luciferase-expressing MDA-MB-231 human breast cancer xenografts wereorthotopically implanted in the mammary fat pads of female nude (nu/nu)mice. When the average tumor volume reached 230 mm³, mice wererandomized into groups of five animals and treated with saline,Abraxane®, Avastin®, or a combination of Abraxane® plus Avastin®.Abraxane®, either alone or in combination, was administered at 10mg/kg/day daily for 5 days in two cycles separated by 1 week. Avastin®was administered following the two cycles of Abraxane® at dosages of 2mg/kg, 4 mg/kg or 8 mg/kg twice a week for 6 weeks. Avastin® alone wasadministered at a dosage of 4 mg/kg at the same time as mice incombination therapy. Mice were monitored for tumor growth and drugtoxicity. Mice were sacrificed when the mean tumor volume in thesaline-treated control group reached 2000 mm³.

TABLE 5 Mean Avastin ® Tumor Volume % Complete Treatment Dose (mm³) %TGI Regression³ Saline control 2391 ± 432 0 Abraxane ® 117 ± 38 95.11 0(ABX) Avastin ® 4 mg/kg 2089 ± 251 12.56 0 ABX + 2 mg/kg 138 ± 42 94.2320 (1/5) Avastin ® ABX + 4 mg/kg  60 ± 17 97.49 40 (2/5) Avastin ® ABX +8 mg/kg  36 ± 16 98.49 40 (2/5) Avastin ®

No toxicity was observed in any treatment group. Tumor growth inhibition(TGI) was calculated by comparing mean tumor volume of test groups tothat of the control group at the last measurement of the control group.As shown in Table 5 and in FIG. 12, Avastin® at a dose of 4 mg/kg didnot significantly inhibit growth of primary tumors (12.56% inhibition).Abraxane and Avastin® combination therapy yielded a significantly betteroutcome than Avastin® alone, with tumor inhibition ranging from 94.23%to 98.49%. Abraxane® in combination with Avastin® at the two highestdoses, yielded a better outcome that Abraxane® alone (97.49 or 98.49%compared to 95.11% inhibition). Abraxane® and Avastin® in combinationresulted in regression of tumors in treated mice wherein completeregression referred to mice with no measurable tumors at day 65. Five offifteen (30%) mice treated with a combination of Abraxane® and

Avastin® showed complete tumor regression; tumors in the remaining micewere reduced by 90% compared with controls.

Example 27 Abraxane® (ABI-007) in Combination with Avastin® ReducesTumor Metastasis in MDA-MB-231 Tumor Xenografts

As described in Example 25, luciferase-expressing MDA-MB-231 humanbreast cancer xenografts were orthotopically implanted in the mammaryfat pads of female nude (nu/nu) mice. When the average tumor volumereached 230 mm³, mice were randomized into groups and treated withsaline (n=10), Abraxane® (n=5), Avastin® (n=5), or a combination ofAbraxane® plus Avastin® (n=5). Abraxane®, either alone or incombination, was administered at 10 mg/kg/day daily for 5 days in twocycles separated by 1 week. Avastin® was administered following the twocycles of Abraxane® at dosages of 2 mg/kg, 4 mg/kg or 8 mg/kg twice aweek for 6 weeks. Avastin® alone was administered at a dosage of 4 mg/kgat the same time as mice in combination therapy. Mice were sacrificedwhen the mean tumor volume in the saline-treated control group reached2000 mm³. Axillary lymph nodes and both lobes of the lungs were removedfrom each mouse and cellular extracts were prepared. The presence ofMDA-MB-231 cells in these tissues was evaluated by analysis ofluciferase activity and was an indicator of metastasis from the primarytumor. Luciferase activity was measured in extracts from 10 lymph nodesand both lobes of the lungs on the day of sacrifice (day 65 after tumorimplantation). A value greater than 500 light units per 20 PI lysate wasrated as positive for presence of MDA-MB-231 cells and for incidence ofmetastasis.

TABLE 6 Lymph Node Pulmonary Metastasis Metastasis Avastin ® P PTreatment Dose Incidence Value Incidence Value Saline 10/10 (100%)  7/10(70%) control Abraxane ® 5/5 (100%) — 4/5 (80%) — (ABX) Avastin ® 4mg/kg 5/5 (100%) — 3/5 (60%) NS ABX + 2 mg/kg 5/5 (100%) — 1/5 (20%)0.045 Avastin ® ABX + 4 mg/kg 2/5 (40%)  0.022 2/5 (40%) NS Avastin ®ABX + 8 mg/kg 2/5 (40%)  0.022 0/5 (0%)  0.025 Avastin ®

As shown in Table 6, treatment with Abraxane® or Avastin® alone appearedto have no effect on the incidence of tumor metastasis to the lymphnodes as analyzed by luciferase activity in cellular extracts. As usedherein, incidence refers to the presence of luciferase activity intissue from each mouse. Abraxane® in combination with Avastin® diddemonstrate a significant effect on tumor metastasis. Incidence ofmetastases fell to 40% in groups treated with Abraxane® and Avastin® atthe two highest dosages of 4 mg/kg and 8 mg/kg. (P value=0.022; whereinthe P value was generated by the analysis of difference between test andcontrol groups with Fisher's exact test; NS refers to non-significant.)Abraxane or Avastin® alone appeared to have little effect on incidenceof tumor metastasis to the lungs as shown in Table 6. Abraxane® incombination with Avastin® demonstrated an effect on the incidence oflung metastases. Incidence of metastases fell to 20%, 40% and 0% withcombinations of Abraxane® and Avastin® at dosages of 2 mg/kg, 4 mg/kgand 8 mg/kg, respectively.

Tumor metastasis to the lymph nodes and lungs as evaluated by luciferaseactivity in tissue extracts is shown in FIG. 13. The combination ofAbraxane® and Avastin® had a synergistic effect on reducing lymph nodemetastases (FIG. 13A) and lung metastases (FIG. 13B) of the MDA-MB-231tumor cells.

Example 28 Paclitaxel Chemotherapy Increases Microvessel Density inTumors

Solvent-based (i.e., Taxol®) and solvent-free (i.e., nab-paclitaxel,Abraxane®) formulations of paclitaxel were assayed for their ability toreduce tumor volume and to increase microvessel density (“MVD”) intumors. Parallel experiments each containing four groups of five micebearing either paclitaxel-sensitive MX-1 xenografts,paclitaxel-sensitive MES-SA xenografts, or paclitaxel-resistantMES-SA/Dx5 xenografts were treated with: (1) Taxol® at 13.4 mg/kgadministered once daily for five consecutive days (“qdx5”); (2)Abraxane® at 13.4 mg/kg qdx5; (3) Abraxane® at 30 mg/kg qdx5; or (4) acomparable volume of phosphate-buffered saline (“PBS”) qdx5. In oneexperiment, tumor volume was assessed twice weekly starting at day 17.In the second experiment, MVD was quantified by CD31 staining at day 11,the last day of the experiment. MVD was reported as percent ofCD31-positive structures in each tissue relative to tumor volume.

As shown in FIG. 14A, tumor growth inhibition confirmed that MES-SA/Dx5is paclitaxel-resistant and that MX-1 and MES-SA arepaclitaxel-sensitive. As shown in FIG. 14B, the MVD increased with tumorshrinkage. For MX-1, MVD increased from 1.08±0.65% (PBS) to 4.93±3.22%(Taxol® at 13.4 mg/kg), 9.03±13.0% (Abraxane® at 13.4 mg/kg), and9.18±11.19% (Abraxane® at 30 mg/kg). For MES-SA, MVD increased from3.96+3.68% (PBS), to 7.33+1.30 (Taxol® at 13.4 mg/kg), 3.33+1.03%(Abraxane® at 13.4 mg/kg), and 11.69+7.51% (Abraxane® at 30 mg/kg). ForMES-SA/Dx5, MVD remained stable at 4.16+2.39%, 4.11+0.55% (Taxol® at13.4 mg/kg), 4.13+2.30% (Abraxane® at 13.4 mg/kg), and 2.52+1.08%(Abraxane® at 30 mg/kg). In both paclitaxel-sensitive MX-1 and MES-SA,there was a positive correlation between decreasing tumor volume andincreasing MVD (compare MX-1 panels in FIGS. 14A and 14B and MES-SApanels in FIGS. 14A and 14B). There was no observable change in tumorvolume and MVD in paclitaxel-resistant MES-SA/Dx5 after treatment withTaxol® at 13.4 mg/kg, with either concentration of Abraxane®, or withPBS. The fourth panel of FIG. 14B shows the MVD data for all three tumortypes plotted together. That data indicated that tumors undergoingpaclitaxel-induced regression exhibited increased MVD, reflectingincreased angiogenesis in response to chemotherapy, and that therelationship was log linear.

Example 29 Administration of Avastin® in Combination with Abraxane®(ABI-007) Significantly Improves Tumor Suppression Induced by Abraxane®(ABI-007) Alone

The anti-tumor activity of Abraxane® alone and in combination withbevacizumab (Avastin®) was assayed in vivo. MDA-MB-231-Luc+human breastcancer xenografts were orthotopically implanted in the mammary fat pads(“MFP”) of 4 to 6 week old female nude (nu/nu) mice (HarlanSprague-Delaney, Indianapolis, Ind.), according to standard methods.Mice bearing MDA-MB-231 tumors of 200-250 mm in volume were randomizedinto 6 groups of 10 animals each and treated with: (1) PBS; (2)Abraxane® alone (10 mg/kg, administered intravenously (“i.v.”) qdx5);(3) Avastin® alone (4 mg/kg, administered by intraperitoneal (“i.p.”)injection three times weekly (“q3xwkly”)); or (4) Abraxane® followed byAvastin®. Avastin® treatment began 24 hours after the end of one cycleof Abraxane®, at a dose of 2 mg/kg, 4 mg/kg or 8 mg/kg of Avastin®dissolved in 0.1 mL of sterile saline and injected intraperitoneallyq3xwkly for 5 weeks. One or two cycles of Abraxane® were administered.In two-cycle Abraxane® treatment, a second cycle of Abraxane® (10 mg/kg,i.v. qdx5) was administered one week after conclusion of the firstcycle. The control group received saline administered in the same volumeby the same method (i.e., 0.1 ml injected i.v. or i.p.) on the same daysas Abraxane® and Avastin® treatments, respectively.

Two rounds of experiments were performed on mice bearingluciferase-expressing MDA-MS-231-derived tumors to compare the efficacyof one or two qdx5 cycles of Abraxane® chemotherapy combined withAvastin®. No signs of toxicity as judged by weight loss or behavioralchanges were observed in any of the treated mice. A comparative analysisof both experiments presented in FIG. 15 and Table 7 showed that twocycles of Abraxane® (10 mg/kg) were significantly more effective insuppressing tumor growth than one cycle (p<0.05). FIG. 15 presents datafrom combination therapy with Abraxane® at 10 mg/kg and Avastin® at 4mg/kg.

TABLE 7 Mean Tumor Volume % Complete Treatment Avastin ® Dose (mm³)¹ %TGI² % TGD³ Regression⁴ Experiment No. 1: One cycle of Abraxane ® (10mg/kg) Saline control 2079 ± 287 0 Abraxane ® (ABX) 596 ± 98 71.33 20 0Avastin ® 2 mg/kg  953 ± 127 54.16 13 0 ABX + Avastin ® 2 mg/kg 255 ± 4687.73 33 0 Experiment No. 2: Two cycles of Abraxane ® (10 mg/kg) Salinecontrol 2391 ± 432 0 Abraxane ® (ABX) 117 ± 38 95.11 >65⁵ 0 Avastin ® 4mg/kg 2089 ± 251 12.56  7 0 ABX + Avastin ® 2 mg/kg 138 ± 42 94.23>65^(e) 20 (1/5) ABX + Avastin ® 4 mg/kg  60 ± 17 97.49 >65^(e) 40 (2/5)ABX + Avastin ® 8 mg/kg  36 ± 16 98.49 >65^(e) 40 (2/5) ¹The mean tumorvolume per group (n = 5) ± SE at day of sacrifice of PBS-treated controlgroup because their tumors reached a 2000 mm³ limit. ²Percent reductionin the mean tumor volume in drug-treated groups as compared with that inPBS-treated group at the last day of measurement of control mice. ³Tumorgrowth delay (“TGD”) indicates the number of additional days requiredfor averaged tumor volume in experimental groups to reach 1000 mm³compared to that in PBS-treated control group. The control group reached1000 mm³ at day 25 post tumor implantation. ⁴Percent of mice with nomeasurable tumors at day 65 post tumor implantation. Numbers inparentheses indicate a number of mice with complete regressed tumors ineach group. ⁵TGD could not be accurately determined because the meantumor volume did not reach 1000 mm³ by the end of the experiment (day 65post tumor implantation).

As shown in Table 7, the mean tumor volume after one cycle of Abraxane®was 596±98 mm³, corresponding to 71% TGI compared to the average tumorvolume in the control group, while the mean tumor volume after twocycles of Abraxane® was 117±38 mm³, corresponding to 95% TGI comparedwith the average tumor volume in the control group. One cycle ofAbraxane® delayed the time point at which tumor volume reached 1000 mm³by 20 days relative to control animals. In contrast, the mean tumorvolume in the group receiving two cycles of Abraxane® did not reach 1000mm³ over the entire duration of the experiment, thus prolonging tumorgrowth delay (“TGD”) for a minimum of 65 days. Avastin® alone (4 mg/kg)produced minor (FIG. 15A) to no significant effect (FIG. 15B) on tumorgrowth, since the experiments were performed in mice withwell-established tumors (100-150 mm³ at the first day of treatment).

Furthermore, the results demonstrated that combination therapy withAbraxane® and Avastin® delivered a synergistic suppression of tumorgrowth compared with the either drug administered alone. Even thoughAbraxane® alone potently inhibited tumor growth, particularly with twocycle therapy, tumor growth resumed in all mice regardless of the extentof tumor suppression and tumor growth delay, and no complete regressionswere observed in any of the mice treated with either Abraxane® orAvastin® alone. In contrast, mice receiving combination therapy hadsmaller tumors on average compared with those in any other experimentalgroup (p<0.05). In addition, several mice displayed no measurable orvisually detectable tumors at all (FIG. 15B and Table 7) after receivingtwo cycles of Abraxane® in combination with 2, 4 or 8 mg/kg of Avastin®.Although the relatively small number of mice per group did not allow adirect dose dependency to be established with regard to Avastin®concentrations, there was a trend showing that higher doses of Avastin®appeared to be associated with a higher number of mice with completelyregressed tumors (Table 7).

Example 30 Combination Therapy with Abraxane® (ABI-007) and Avastin® butNot Abraxane® (ABI-007) or Avastin® Alone Resulted in Complete,Sustainable Tumor Regressions in All Treated Mice

Mice bearing MDA-MB-231-derived tumors were generated as described inExample 29. Mice bearing tumors of 200-250 mm³ in volume were randomizedinto 6 groups of 10 animals each and treated with: (1) PBS; (2) Avastin®alone (4 mg/kg, administered by intraperitoneal (“i.p.”) injection twiceweekly (“q2xwkly”)); (3) Abraxane® alone (30 mg/kg, administeredintravenously (“i.v.”) qdx5); or (4) Abraxane® followed by Avastin®.Avastin® treatment began 24 hours after the first injection of Abraxane®and continued until the end of the experiment at a dose of 4 mg/kg ofAvastin® dissolved in 0.1 mL of sterile saline, injectedintraperitoneally q2xwkly for 5 weeks. Two cycles of Abraxane® wereadministered (both at 10 mg/kg, i.v. qdx5), separated by one week. Thecontrol group received saline administered in the same volume by thesame method (i.e., 0.1 ml injected i.v. or i.p.) on the same days asAbraxane® and Avastin® treatments, respectively.

Tumor volume was assessed three times a week until completion of thestudy. A summary of the data is shown in FIG. 16. Notably, while twocycles of Abraxane® (30 mg/kg) alone effectively suppressed tumorgrowth, the tumors began to regrow approximately two weeks after the endof the second cycle of Abraxane®, though somewhat more slowly than inmice treated with Avastin® (4 mg/kg) alone or the PBS-injected controlanimals. In contrast, two cycles of Abraxane® (30 mg/kg) administered incombination with Avastin® (4 mg/kg) not only effectively suppressedtumor growth, but resulted in nearly complete regression of tumorsthrough the end of the experiment, 95 days after tumor implantation.

Example 31 Combination Therapy with Abraxane® (ABI-007) and Avastin®Inhibits Tumor Growth, Lymphatic and Pulmonary Metastasis in Mice withMDA-MB-435-Luc+-Derived Tumors

The luciferase-tagged MDA-MB-435 cell line (“MDA-MB-435-Luc+”) was agenerous gift of Dr. Sierra (Universitaria de Bellvitge, Barcelona,Spain), and has been characterized elsewhere. Rubio, N., et al. (2001),“Metastatic behavior of human breast carcinomas overexpression theBcl-x(L) gene: a role in dormancy and organospecificity,” Lab. Invest.8:725-34. The MDA-MB-435-Luc+ cell line has high metastatic potential,predominantly to the lungs and, to a lesser degree, to lymph nodes. Theculture medium for all tumor lines consisted of Dulbecco's ModifiedEagle Medium supplemented with 5% fetal bovine serum, 2 mM glutamine, 1mM sodium pyruvate and nonessential amino acids. Tumor cells wereharvested for passage by washing the cell monolayer with PBS, followedby 3-4 minutes exposure to 0.5 mM EDTA diluted in PBS. Cells weresub-cultured twice weekly and routinely tested for mycoplasma using animmunodetection kit from Roche Diagnostics GmbH (Penzberg, Germany).

MDA-MB-435-Luc+ (“435-Luc+”) cells were implanted subcutaneously intothe mammary fat pad of 4 to 6 week old female ICR SCID mice (Taconic,Hudson, N.Y.). Every 2-3 days, perpendicular tumor diameters weremeasured by digital caliper and used to calculate tumor volume accordingto the formula: volume=Dd² π/6, where D=larger diameter and d=smallerdiameter. The 435-Luc+tumors had an identical proliferation rate totheir non-transfected counterpart (mice bearing 435-MBA-MS-derivedorthotopic tumors). Animal care was in accordance with institutionalguidelines.

Mice bearing 435-Luc⁺ tumors of 200-250 mm³ in volume were randomizedinto 6 groups of 5 animals each, and treated with PBS, Abraxane® (“ABX”)alone (10 mg/kg, i.v., qdx5), Avastin® alone (4 mg/kg, i.p., twice aweek) or Abraxane® followed by Avastin®. Two cycles of Abraxane®treatment were administered, each for 5 consecutive days with one weekof rest between cycles. Avastin® treatment began 24 hours after the endof one cycle of Abraxane®, with a dose of 4 mg/kg dissolved in 0.1 ml ofsterile, endotoxin-free PBS and injected i.p., twice a week, for totalof 5 weeks. The control group received PBS injected i.v. or i.p. (0.1ml) on the same days as Abraxane® and Avastin® treatments, respectively.To allow maximal development of metastatic foci, mice were sacrificedwhen the average tumor volume in the control group reached 2000 mm³.Table 8 shows the effect of Abraxane®, Avastin®, and combination therapywith Abraxane® and Avastin® on tumor growth in the MDA-MB-435-Luc+ tumormodel.

TABLE 8 MDA-MB-435-Luc+ Avastin ® Tumor Volume % Complete Treatment(mg/kg) (mm³)⁶ % TGI⁷ Regressions⁸ Control 2020 0 ABX alone 369 81.73 0Avastin alone 4 792 60.79 0 ABX + 4 167 91.73 0 Avastin ® ⁶Tumor volumerepresents the mean tumor volume per group, expressed in cubicmillimeter (“mm³”). ⁷Tumor growth inhibition (“TGI”) is presented aspercent reduction in the mean tumor volume in experimental groupscompared with that in PBS-treated control groups. ⁸Complete regressionwas defined as the absence of either measurable or palpable tumor at theoriginal tumor injection site for the entrie length of the experiment,which was 92 days.

Tumor metastasis was determined by measuring luciferase activity intissue extracts derived from 10 axillary lymph nodes (“LN”) and bothlobes of the lungs. Lymph nodes and lungs were excised, washed in PBS,and homogenized in 0.35 ml of cold CCLR buffer (Promega, Madison, Wis.)containing a protease inhibitor cocktail and PMSF (Sigma, Mo.). Celldebris was removed by centrifugation. Protein concentrations of clearedlysates were determined by Bradford assay (Bio-Rad, Hercules, Calif.).Fifty μl of Luciferase Assay Reagent (Promega, Madison, Wis.) was mixedwith 10 μl of cleared lysate and a 10 second average of luminescence wasdetected using a single tube luminometer (Berthold, Germany). CCLRbuffer without tissue homogenates and lymph nodes or lungs of non-tumorbearing mice were assayed for background signal and subtracted from theresults. The net results were expressed as relative light units (“RLU”)normalized per mg of total lysate protein. To assess incidence ofmetastasis, extracts that had a luminescence signal of 800 light unitsabove background (approximately 100 light units) were rated positive.This value was chosen because it was the minimal signal reproduciblydetected above background in metastatic tissues, independently confirmedby immunohistochemical detection (data not shown). Differences in lymphnode and lung metastases were assessed for statistical significance byFisher's exact test using GraphPad InStat (GraphPad Inc., San Diego,Calif.) or SPSS 14.0 (SPSS, Inc., Chicago, Ill.).

The metastases of MDS-MB-435-Luc+tumor cells were identified bymeasuring luciferase activity in tissue extracts from lymph nodes andlungs. Incidence of metastasis is presented in Table 9. In combinationtherapy groups, 435-Luc⁺ tumor-bearing mice received a 4 mg/kg dose ofAvastin®. As expected, 100% of control 435-Luc⁺ tumor-bearing mice hadpulmonary metastases, while only 50% of controls had lymph nodemetastases (Table 9).

TABLE 9 MDA-MB-435-Luc+ Avastin ® Incidence⁹ % Inhibition Treatment(mg/kg) N/total (%) vs. Control P value Lymph node metastasis Control4/8 (50) ABX alone 0/6 (0)  100 0.01 Avastin alone 4 0/6 (0)  100 0.01ABX + 4 0/6 (0)  100 0.01 Avastin ® Pulmonary metastasis Control  7/7(100) ABX alone 5/6 (83) 17 NS¹⁰ Avastin alone 4 4/8 (50) 50 0.01 ABX +4 2/8 (25) 75  0.001 Avastin ® ⁹Luciferase activity was measured inextracts form 10 lymph nodes and both lobes of the lungs from eachmouse. Sample containing a minimum of 500 light units per 20 μl lysatewere rated positive. Lysates from tissues of non-tumor bearing miceproduced a luminescence signal of 100 light units, which was consideredbackground and subtracted from positive values. Results are presented asnumber of mice with lymph node or lung metastasis per total number ofmice with tumors in each experimental group. Percentage of mice withmetastases is shown in parentheses. ¹⁰NS = non-significant. Thedifference between the experimental and control groups does not reachstatistical significance when analyzed by the Fisher's exact test.

Although neither Abraxane® (10 mg/kg) nor Avastin® (4 mg/kg) effectivelysuppressed pulmonary metastasis in 435-Luc+tumor-bearing mice,remarkably, the Abraxane®/Avastin® combination significantly suppressedpulmonary metastasis, and reduced the number of mice with lung tumors by75% compared to the PBS-injected control. (Table 9). In contrast,Abraxane® (10 mg/kg) alone, Avastin® (4 mg/kg) alone, and theAbraxane®/Avastin® combination therapy all suppressed lymph nodemetastasis. These results indicate that combination therapy withAbraxane® and Avastin® could be particularly beneficial for suppressionof both lymph node and pulmonary metastasis.

Example 32 Combination Therapy with Abraxane® (ABI-007) and Avastin®Eradicated Lymphatic and Pulmonary Metastasis in Mice with MDA-MB-231-or MDA-MB-231-Luc+-Derived Tumors

MDA-MB-231 or 231-Luc+ cells were implanted into the MFP of 4 to 6 weekold female nu/nu mice (Harlan Sprague-Delaney, Indianapolis, Ind.) bystandard methods. Briefly, mice were anesthetized and 100 μl of cellsuspension containing 4×10⁶ cells and 50% Matrigel (Sigma, Mo.) wereinjected into the MFP. Every 2-3 days, perpendicular tumor diameterswere measured by digital caliper and used to calculate tumor volumeaccording to the formula: Volume=Dd²π/6, where D=larger diameter andd=smaller diameter. The 231-Luc⁺ tumors had an identical proliferationrate to their non-transfected counterpart. Animal care was in accordancewith institutional guidelines.

Luciferase-expressing orthotopic MDA-MB-231 (231-Luc+) xenografts weregrown to well-established tumors (˜460 mm³) and randomized to fourgroups of N=5, 6 or 7. Groups were treated with: (1) Abraxane® (30mg/kg, qdx5) alone; (2) Avastin® (4 mg/kg, q2xwkly for the duration ofthe experiment) alone; (3) two cycles of Abraxane® (30 mg/kg, qdx5) incombination with Avastin® (4 mg/kg, q2xwkly for the duration); or (4)with an identical volume of PBS.

Metastases of MDA-MB-231-Luc+tumor cells were identified by measuringluciferase activity in tissue extracts from lymph nodes and lungs, asdescribed in Example 31 above. To assess incidence of metastasis,extracts that had a luminescence signal of 500 light units abovebackground (approximately 100 light units) were rated positive. Thisvalue was chosen because it represented the minimal signal which couldbe reproducibly detected above background in tissues in which metastaseswere independently confirmed by immunohistochemical detection of humancells. The net results were expressed as relative light units (RLU)normalized per mg of total lysate protein, and are presented in FIG. 17A(lymphatic metastasis) and FIG. 17B (pulmonary metastasis).

Consistent with the significantly enhanced antitumor effect, theantimetastatic effect was also dramatically improved bynab-paclitaxel/bevacizumab combination in well-established large tumors.While either bevacizumab or nab-paclitaxel alone failed to preventmetastasis to proximal and contralateral lymph nodes and lungs, thelymphatic and pulmonary metastases were completely eliminated by thecombination therapy (FIG. 6 and Table 3), measured by both metastaticburden and incidence.

These results indicate that combination therapy with Abraxane® (30mg/kg) and Avastin® (4 mg/kg) effectively suppressed both lymphatic andpulmonary metastasis in MDA-MB-231-Luc+-derived tumor-bearing mice,remarkably, the Abraxane®/Avastin® combination significantly suppressedpulmonary metastasis, in contrast to treatment with Abraxane® (30 mg/kg)or Avastin® (4 mg/kg) alone. Thus, combination therapy with Abraxane®and Avastin® could be particularly beneficial for prevention of tumorrebound resulting from reactionary angiogenesis, and for suppression ofboth lymph node and pulmonary metastasis. Furthermore, the improvedantitumor efficacy and better safety profile of Abraxane®(nab-paclitaxel) over conventional solvent-based paclitaxel (Taxol®)make it a more promising candidate for combination therapy. Mostsurprisingly and encouragingly, the Abraxane®/Avastin® combination waseven highly effective against well-established large tumors to theextent of total elimination of both primary tumors and metastasis.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is apparent to those skilled in the art that certainminor changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

Preferred variations of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred variations may become apparent to those ofordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of treating a proliferative disease in an individual,comprising administering to the individual: a) an effective amount of acomposition comprising nanoparticles comprising taxane and a carrierprotein, and b) an effective amount of an anti-VEGF antibody.
 2. Themethod of claim 1, wherein the effective amounts of the nanoparticlecomposition and the anti-VEGF antibody synergistically inhibit cellproliferation.
 3. The method of claim 1, wherein the proliferativedisease is cancer.
 4. The method of claim 3, wherein the cancer isbreast cancer.
 5. The method of claim 1, wherein the anti-VEGF antibodyis bevacizumab.
 6. The method of claim 1, wherein the effective amountof the anti-VEGF antibody is at least about 2 mg/kg.
 7. The method ofclaim 6, wherein the effective amount of the anti-VEGF antibody is atleast about 4 mg/kg.
 8. The method of claim 1, wherein the amount oftaxane in the nanoparticle composition is at least about 10 mg/kg. 9.The method of claim 1, wherein the nanoparticle composition and theanti-VEGF antibody are administered sequentially to the individual. 10.The method of claim 1, wherein the nanoparticle composition isadministered for at least one cycles prior to the administration of theanti-VEGF antibody.
 11. The method of claim 10, wherein theadministration of the nanoparticle composition is followed by theadministration of an anti-VEGF antibody for at least about 3 weeks. 12.The method of claim 1, wherein the taxane is paclitaxel.
 13. The methodof claim 1, wherein the average diameter of the nanoparticles in thecomposition is no greater than about 200 nm.
 14. The method of claim 1,wherein the carrier protein is albumin.
 15. The method of claim 14,wherein the weight ratio of the albumin and the taxane in thenanoparticle composition is less than about 9:1.
 16. The method of claim1, wherein the nanoparticle composition is free of Cremophor.
 17. Themethod of claim 1, wherein the individual is human.
 18. A method ofinhibiting tumor metastasis in an individual, comprising administeringto the individual: a) an effective amount of a composition comprisingnanoparticles comprising taxane and a carrier protein, and b) aneffective amount of an anti-VEGF antibody.
 19. The method of claim 18,wherein the effective amounts of the nanoparticle composition and theanti-VEGF antibody synergistically inhibit tumor metastasis.
 20. Themethod of claim 18, wherein the tumor metastasis is metastasis to lymphnode.
 21. The method of claim 18, wherein the tumor metastasis ismetastasis to the lung.
 22. The method of claim 18, wherein the tumormetastasis is metastasis of breast cancer.
 23. The method of claim 18,wherein at least about 40% of metastasis is inhibited.
 24. The method ofclaim 18, wherein at least about 80% of metastasis is inhibited.
 25. Akit comprising: a) a composition comprising nanoparticles comprising ataxane and a carrier protein, b) an anti-VEGF antibody, and c)instructions for administering the nanoparticles and thechemotherapeutic agents for treatment of a proliferative disease.